Caspase inhibitors and uses thereof

ABSTRACT

The present invention relates to compounds of formula I: 
     
       
         
         
             
             
         
       
     
     useful as inhibitors of caspases. The present invention also provides pharmaceutically acceptable compositions comprising said compounds, processes for preparing the compounds, and methods of using the compounds and compositions in the treatment of various diseases, conditions, or disorders.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application60/392,592 filed Jun. 28, 2002 and U.S. Provisional Application60/435,073, filed Dec. 20, 2002, the entirety of both documents beingincorporated herein by reference.

FIELD OF THE INVENTION

This invention is in the field of medicinal chemistry and relates tocompounds, and pharmaceutical compositions thereof, that inhibitcaspases that mediate cell apoptosis and inflammation. The inventionalso relates to processes for preparing the compounds and to methods ofusing the compounds and pharmaceutical compositions of this invention totreat diseases where caspase activity is implicated.

BACKGROUND OF THE INVENTION

Apoptosis, or programmed cell death, is a principal mechanism by whichorganisms eliminate unwanted cells. The deregulation of apoptosis,either excessive apoptosis or the failure to undergo it, has beenimplicated in a number of diseases such as cancer, acute inflammatoryand autoimmune disorders, ischemic diseases and certainneurodegenerative disorders (see generally Science, 281, 1283-1312(1998); Ellis et al., Ann. Rev. Cell. Biol., 7, 663 (1991)).

Caspases are a family of cysteine protease enzymes that are keymediators in the signaling pathways for apoptosis and cell disassembly(Thornberry, Chem. Biol., 5, R97-R103 (1998)). These signaling pathwaysvary depending on cell type and stimulus, but all apoptosis pathwaysappear to converge at a common effector pathway leading to proteolysisof key proteins. Caspases are involved in both the effector phase of thesignaling pathway and further upstream at its initiation. The upstreamcaspases involved in initiation events become activated and in turnactivate other caspases that are involved in the later phases ofapoptosis.

Caspase-1, the first identified caspase, is also known as interleukinconverting enzyme or “ICE.” Caspase-1 converts precursor interleukin-1 β(“pIL-1β”) to the pro-inflammatory active form by specific cleavage ofpIL-1β between Asp-116 and Ala-117. Besides caspase-1 there are alsoeleven other known human caspases, all of which cleave specifically ataspartyl residues. They are also observed to have stringent requirementsfor at least four amino acid residues on the N-terminal side of thecleavage site.

The caspases have been classified into three groups depending on theamino acid sequence that is preferred or primarily recognized. The groupof caspases, which includes caspases 1, 4, 5 and 13, have been shown toprefer hydrophobic aromatic amino acids at position 4 on the N-terminalside of the cleavage site. Another group that includes caspases 2, 3 and7, recognize aspartyl residues at both positions 1 and 4 on theN-terminal side of the cleavage site, and preferably a sequence ofAsp-Glu-X-Asp. A third group, which includes caspases 6, 8, 9 and 10,tolerate many amino acids in the primary recognition sequence, but seemto prefer residues with branched, aliphatic side chains such as valineand leucine at position 4.

The caspases have also been grouped according to their perceivedfunction. The first subfamily consists of caspases-1 (ICE), 4, 5 and 13.These caspases have been shown to be involved in pro-inflammatorycytokine processing and therefore play an important role ininflammation. Caspase-1, the most studied enzyme of this class,activates the IL-1β precursor by proteolytic cleavage. This enzymetherefore plays a key role in the inflammatory response. Caspase-1 isalso involved in the processing of interferon-γ inducing factor (IGIF)which stimulates the production of interferon gamma, a keyimmunoregulator that modulates antigen presentation, T-cell activationand cell adhesion.

The remaining caspases make up the second and third subfamilies. Theseenzymes are of central importance in the intracellular signalingpathways leading to apoptosis. One subfamily consists of the enzymesinvolved in initiating events in the apoptotic pathway, includingtransduction of signals from the plasma membrane. Members of thissubfamily include caspases-2, 8, 9 and 10. The other subfamily,consisting of the effector caspases 3, 6 and 7, are involved in thefinal downstream cleavage events that result in the systematic breakdownand death of the cell by apoptosis. Caspases involved in the upstreamsignal transduction activate the downstream caspases, which then disableDNA repair mechanisms, fragment DNA, dismantle the cell cytoskeleton andfinally fragment the cell.

Knowledge of the four amino acid sequence primarily recognized by thecaspases has been used to design caspase inhibitors. Reversibletetrapeptide inhibitors have been prepared having the structureCH₃CO—[P4]-[P3]-[P2]-CH(R)CH₂CO₂H where P2 to P4 represent an optimalamino acid recognition sequence and R is an aldehyde, nitrile or ketonecapable of binding to the caspase cysteine sulfhydryl. Rano andThornberry, Chem. Biol. 4, 149-155 (1997); Mjalli, et al., Bioorg. Med.Chem. Lett. 3, 2689-2692 (1993); Nicholson et al., Nature 376, 37-43(1995). Irreversible inhibitors based on the analogous tetrapeptiderecognition sequence have been prepared where R is anacyloxymethylketone —COCH₂OCOR′. R′ is exemplified by an optionallysubstituted phenyl such as 2,6-dichlorobenzoyloxy and where R is COCH₂Xwhere X is a leaving group such as F or Cl. Thornberry et al.,Biochemistry 33, 3934 (1994); Dolle et al., J. Med. Chem. 37, 563-564(1994).

The utility of caspase inhibitors to treat a variety of mammaliandisease states associated with an increase in cellular apoptosis hasbeen demonstrated using peptidic caspase inhibitors. For example, inrodent models caspase inhibitors have been shown to reduce infarct sizeand inhibit cardiomyocyte apoptosis after myocardial infarction, toreduce lesion volume and neurological deficit resulting from stroke, toreduce post-traumatic apoptosis and neurological deficit in traumaticbrain injury, to be effective in treating fulminant liver destruction,and to improved survival after endotoxic shock. Yaoita et al.,Circulation, 97, 276 (1998); Endres et al., J Cerebral Blood Flow andMetabolism, 18, 238, (1998); Cheng et al., J. Clin. Invest., 101, 1992(1998); Yakovlev et al., J Neuroscience, 17, 7415 (1997); Rodriquez etal., J. Exp. Med., 184, 2067 (1996); Grobmyer et al., Mol. Med., 5, 585(1999).

In general, the peptidic inhibitors described above are very potentagainst some of the caspase enzymes. However, this potency has notalways been reflected in cellular models of apoptosis. In additionpeptide inhibitors are typically characterized by undesirablepharmacological properties such as poor oral absorption, poor stabilityand rapid metabolism. Plattner and Norbeck, in Drug DiscoveryTechnologies, Clark and Moos, Eds. (Ellis Horwood, Chichester, England,1990).

Recognizing the need to improve the pharmacological properties of thepeptidic caspase inhibitors, peptidomimetic inhibitors have beenreported. Amongst these, inhibitors where the P3 amino acid has beenreplaced by derivatives of 3-aminopyridin-2-ones and5-aminopyrimidin-4-ones have received much attention (U.S. Pat. No.5,756,466 (Bemis et al.); Dolle et al. J. Med. Chem. 39, 2438, (1996);Golec et al. Bioorg. Med. Chem. Lett. 7, 2181, (1997); Semple et al,Biorg. Med. Chem. Lett. 7, 1337, (1997) involving compounds of generalstructure:

wherein R₁-R₄ and X are various groups.

Due to the inherent problems of the peptidic inhibitors, there continuesto be a need for small molecule, nonpeptide caspase inhibitors that arepotent, stable, and penetrate membranes to provide effective inhibitionof apoptosis in vivo. Such compounds would be extremely useful intreating the aforementioned diseases where caspase enzymes play a role.WO 01/42216 discloses caspase inhibitors and uses thereof. The presentinvention provides a selection over the WO 01/42216 disclosure.

SUMMARY OF THE INVENTION

The present invention provides a compound of formula I:

wherein:

X is CH or N;

Y is halo, trifluorophenoxy, or tetrafluorophenoxy;R² is a C₁₋₆ straight chained or branched alkyl;R³ is hydrogen, halo, OCF₃, CN, or CF₃; andR⁴ is hydrogen, halo, OCF₃, SR, CN, CF₃, Ar, or T-Ar;

wherein:

-   -   T is O or S;    -   R is a C₁₋₆ straight chained or branched alkyl;    -   Ar is a phenyl ring optionally substituted with 1-3 groups        selected from halo, CH₃, CF₃, CN, OMe, OCF₃, and NR⁵R⁶; and    -   R⁵ and R⁶ each is independently H or C₁₋₆ alkyl, or R⁵ and R⁶,        taken together, form a 5-7 membered ring optionally containing        up to 3 heteroatoms selected from O, S, NH, and N(C₁₋₆ alkyl);        provided that when Y is halo, then both, R³ and R⁴, are not        simultaneously hydrogen.

The present invention also provides pharmaceutical compositions andmethods using such compositions for treating a caspase-mediated disease.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a compound of formula I:

wherein:

X is CH or N;

Y is halo, trifluorophenoxy, or tetrafluorophenoxy;R² is a C₁₋₆ straight chained or branched alkyl;R³ is hydrogen, halo, OCF₃, CN, or CF₃; andR⁴ is hydrogen, halo, OCF₃, SR, CN, CF₃, Ar, or T-Ar;

wherein:

T is O or S;

R is a C₁₋₆ straight chained or branched alkyl;

Ar is a phenyl ring optionally substituted with 1-3 groups selected fromhalo, CH₃, CF₃, CN, OMe, OCF₃, and NR⁵R⁶; and

R⁵ and R⁶ each is independently H or C₁₋₆ straight chained or branchedalkyl, or R⁵ and R⁶, taken together, form a 5-7 membered ring optionallycontaining up to 3 heteroatoms selected from O, S, NH, andN(C₁₋₆-straight chained or branched alkyl);

provided that when Y is halo, then both, R³ and R⁴, are notsimultaneously hydrogen.

In another embodiment of formula I, R⁴ is hydrogen, halo, OCF₃, CN, CF₃,or T-Ar; wherein T, Ar, and the other variables are as defined above.

The compounds of the present invention represent a selection over thegenus of International PCT Publication WO 01/42216, the entiredisclosure of which is incorporated herein. Specifically, the compoundsof the present invention have an unexpected and surprising ability toinhibit apoptosis and/or inhibit Il-1β release from activated cells.

According to a preferred embodiment, R² in formula I is ethyl, n-propyl,or isopropyl.

According to a more preferred embodiment, R² in formula I is ethyl.

According to an even more preferred embodiment, R² in formula I is(S)-ethyl.

According to a preferred embodiment, R³ in formula I is hydrogen.

According to a preferred embodiment, Y in formula I is F,trifluorophenoxy, or tetrafluorophenoxy.

According to another preferred embodiment, the present inventionprovides a compound of formula IA:

wherein:

R² is ethyl, n-propyl, or isopropyl; and

R³ and R⁴ are each independently hydrogen, halo, OCF₃, CN, CF₃, or Ar,provided that both R³ and R⁴ are not simultaneously hydrogen;

Ar is a phenyl ring optionally substituted with 1-3 groups selected fromhalo, CH₃, CF₃, CN, OMe, OCF₃, and NR⁵R⁶; and

R⁵ and R⁶ each is independently H or C₁₋₆ straight chained or branchedalkyl, or R⁵ and R⁶, taken together, form a 5-7 membered ring optionallycontaining up to 3 heteroatoms selected from O, S, NH, andN(C₁₋₆-straight chained or branched alkyl);

According to another embodiment of formula IA, R³ and R⁴ are eachindependently hydrogen, halo, OCF₃, CN, or CF₃, provided that both, R³and R⁴, are not simultaneously hydrogen; and R² is as defined above forformula IA.

According to a preferred embodiment, R² in the compound of formula IA isethyl.

According to a more preferred embodiment, R² in formula IA is (S)-ethyl.

According to another preferred embodiment, R² in the compound of formulaIA is isopropyl.

According to a preferred embodiment, R³ in the compound of formula IA ishydrogen.

According to a preferred embodiment, R⁴ in the compound of formula IA isF, Cl, CN, Ar, or CF₃.

According to a more preferred embodiment, R⁴ in the compound of formulaIA is Cl.

According to another more preferred embodiment, R⁴ in the compound offormula IA is CF₃.

According to another preferred embodiment, R³ in the compound of formulaIA is hydrogen, and R⁴ is F, Cl, CN, Ar, or CF₃.

According to another preferred embodiment, R³ in the compound of formulaIA is hydrogen and R⁴ is Cl.

According to another preferred embodiment, R³ in the compound of formulaIA is hydrogen and R⁴ is CF₃.

According to another preferred embodiment, R³ in the compound of formulaIA is hydrogen and R⁴ is Ar.

According to another preferred embodiment, the present inventionprovides a compound of formula IB:

wherein:

-   -   X is CH or N;    -   R² is ethyl, n-propyl, or isopropyl;    -   R³ and R⁴ are each independently hydrogen, halo, OCF₃, CN, or        CF₃; and    -   Ar is trifluorophenyl or tetrafluorophenyl.

According to a preferred embodiment, Ar² in the compound of formula IBis 2,3,5,6-tetrafluorophenyl.

According to a preferred embodiment, R² in the compound of formula IB isethyl.

According to a more preferred embodiment, R² in formula IB is (S)-ethyl.

According to a preferred embodiment, X in the compound of formula IB isCH.

According to a preferred embodiment, R³ in the compound of formula IB ishydrogen.

According to a preferred embodiment, R⁴ in the compound of formula IB isF, Cl, or CF₃.

According to a more preferred embodiment, R⁴ in the compound of formulaIB is Cl.

According to a more preferred embodiment, R⁴ in the compound of formulaIB is CF₃.

According to a preferred embodiment, R³ in the compound of formula IB ishydrogen, and R⁴ is F, Cl, or CF₃.

According to another preferred embodiment, Ar² in the compound offormula IB is 2,3,5,6-tetrafluorophenyl, R³ is hydrogen, and R⁴ is Cl.

According to another preferred embodiment, the present inventionprovides a compound of formula IC:

wherein:

-   -   R² is ethyl, n-propyl, or isopropyl;    -   R³ is hydrogen, halo, OCF₃, CN, or CF₃;    -   R⁴ is halo, OCF₃ CN, CF₃, SR, or T-Ar;    -   T is O or S;    -   R is a C₁₋₆ straight chained or branched alkyl;    -   Ar is a phenyl ring optionally substituted with 1-3 groups        selected from halo, CH₃, CF₃, CN, OMe, OCF₃, and NR⁵R⁶; and    -   R⁵ and R⁶ each is independently H or C₁₋₆ straight chained or        branched alkyl, or R⁵ and R⁶, taken together, form a 5-7        membered ring optionally containing up to 3 heteroatoms selected        from O, S, NH, and N(C₁₋₆-straight chained or branched alkyl).

According to a preferred embodiment of the compound of formula IC, R⁴ isT-Ar;

According to a preferred embodiment, R² in the compound of formula IC isethyl.

According to a more preferred embodiment, R² in formula IC is (S)-ethyl.

According to a preferred embodiment, R³ in the compound of formula IC ishydrogen.

According to a preferred embodiment, R⁴ in the compound of formula IC isF, Cl, CN, or CF₃.

According to a more preferred embodiment, R⁴ in the compound of formulaIC is Cl.

According to a preferred embodiment, T in the compound of formula IC isO.

According to a preferred embodiment, Ar if present in the compound offormula IC, is optionally substituted with CF₃ or halo (preferably, thehalo is chloro or fluoro).

As used herein, “C₁₋₆” indicates the presence of 1, 2, 3, 4, 5, or 6carbon atoms (or optional heteroatom substituted therefor).

According to a more preferred embodiment, the present invention providesa compound of formula I, selected from Table 1 below:

TABLE 1 (I)

Example R⁴ R³ R² Y X 1 Cl H Et F CH 2 CF₃ H Et F CH 3 H Cl Et F CH 4 HCF₃ Et F CH 5 Cl H Et 2,3,5,6- N tetrafluoro phenoxy 6 H H Et 2,3,5,6- Ntetrafluoro phenoxy 7 H H Et 2,4,6- N trifluoroph enoxy 8 H H Et 2,3,6-N trifluoroph enoxy 9 H H Et 2,3,5,6- CH tetrafluoro phenoxy 10 Cl H Et2,3,5,6- CH tetrafluoro phenoxy 11 Cl Cl Et 2,3,5,6- CH tetrafluorophenoxy 12 2-chloro- H Et F N phenoxy 13 3-chloro- H Et F N phenoxy 143-fluoro- H Et F N phenoxy 15 2,4- H Et F N dichloro- phenoxy 16phenylsul H Et F N fanyl 17 3-fluoro- H Et F N phenylsul fanyl 18 Cl Hi-Pr F CH 19 CF H Et 2,3,5,6- CH tetrafluoro phenoxy 20 CF H Et 2,3,5,6-N tetrafluoro phenoxy 21 phenyl H Et F CH 22 S(n-Pr) H Et F N

According to another embodiment, the present invention provides apharmaceutical composition comprising:

a) a compound of formula I, as defined above, or a pharmaceuticallyacceptable salt thereof; and

b) a pharmaceutically acceptable carrier, adjuvant or vehicle.

According to a preferred embodiment, the pharmaceutical composition ofthe present invention comprises:

a) a compound of formula IA, formula IB, or formula IC; and

b) a pharmaceutically acceptable carrier, adjuvant or vehicle.

According to a more preferred embodiment, the pharmaceutical compositionof the present invention comprises a compound selected from Table 1above.

Compounds of this invention may exist in solution as either the openform 1 or the cyclized hemiketal form 2. The representation herein ofeither isomeric form is meant to include the other.

It will be apparent to one skilled in the art that certain compounds ofthis invention may exist in tautomeric forms or hydrated forms, all suchforms of the compounds being within the scope of the invention. Unlessotherwise stated, structures depicted herein are also meant to includeall stereochemical forms of the structure; i.e., the R and Sconfigurations for each asymmetric center. Therefore, singlestereochemical isomers as well as enantiomeric and diastereomericmixtures of the present compounds are within the scope of the invention.Unless otherwise stated, structures depicted herein are also meant toinclude compounds that differ only in the presence of one or moreisotopically enriched atoms. For example, compounds having the presentstructures except for the replacement of a hydrogen by a deuterium ortritium, or the replacement of a carbon by a ¹³C- or ¹⁴C-enriched carbonare within the scope of this invention.

The compounds of this invention may be prepared in general by methodsknown to those skilled in the art for analogous compounds. Syntheticroutes to the compounds of the present invention are genericallydescribed in WO 01/42216. For the purposes of illustration, thefollowing Schemes I-IV for the synthesis of the compounds of the presentinvention are provided.

Reagents: (a) EDC/DMAP/HOBt/THF; (b) Dess-Martin periodinane; (c)TFA/DCM

In Scheme I above, the following abbreviations are used: EDC is1-(3-dimethylaminopropyl)-3-ethylcarbodiimide; HOBt is1-hydroxybenzotriazole; THF is tetrahydrofuran; TFA is trifluoroaceticacid; DCM is dichloromethane; DMAP is 4-dimethylaminopyridine. Acid A iscoupled to amino alcohol B to give amide C. The hydroxy group incompound C is oxidized to give the ketone followed by acid hydrolysis ofthe tert butyl ester to give I′ as the free acid.

In the case of fluoromethyl ketones where CH₂Y is CH₂F, the aminoalcohol B may be obtained according to the method of Revesz et al.,Tetrahedron Lett. 35, 9693 (1994). In the case of fluoro-substitutedphenoxy ketones where Y in CH₂Y is 2,3,5,6-tetrafluorophenoxy,2,4,6-trifluorophenoxy, or 2,3,6-trifluorophenoxy, the amino alcohol Bmay be obtained by methods analogous to those of Semple et al.,Bioorganic and Medicinal Chemistry Letters, 7, 1337 (1997) (Scheme II).

Reagents: (a) KF/DMF/Ar²OH; (b) NaBH₄/THF; (c) H₂/Pd/C/MeOH

In scheme II above, the following abbreviations are used: KF ispotassium fluoride; DMF is N,N-dimethylformamide; Ar²OH is either2,3,5,6-tetrafluorophenol, 2,4,6-trifluorophenol or2,3,6-trifluorophenol; THF is tetahydrofuran; MeOH is methanol.Commercially available bromoketone D is reacted with the appropriatelysubstituted fluorophenol and potassium fluoride to give phenoxy ketoneE. The ketone is then reduced with sodium borohydride to give thealcohol F, which is hydrogenated using palladium on carbon as catalystto give the amino alcohol B′ (Ar²=fluoro-substituted phenyl).

Reagents: (a) heat; (b) conc. HCl/IPA; (c) TFA/DCM

In Scheme III above, the following abbreviations are used: IPA isisopropyl alcohol; TFA is trifluoroacetic acid and DCM isdichloromethane. Isoquinolin-1-one acid derivatives (in formula I, X═C)can be prepared in chiral form using the synthetic sequence shown inScheme III. The starting isocoumarin G is prepared by methods analogousto Narasimhan et al., Synthesis, 797 (1975) and Margaretha et al.,Tetrahedron 56, 6763 (2000) unless stated otherwise. Isocoumarin G isfirst heated with commercially available (S)-2-aminobutyric acid,tert-butyl ester. The resulting compound is reacted with concentratedhydrochloric acid in isopropanol to give the isoquinolin-1-onetert-butyl ester which is deprotected to provide the acid H usingtrifluoroacetic acid. The acid is then coupled to amino alcohol B(Scheme I).

Reagents: (a) LiOH/THF/H₂O; (b) (S)-2-Aminobutyric acid, tert-butylester/EDC/HOBt/DMAP/THF; (c) SnCl₂/MeOH; (d)Trimethylorthoformate/AcOH/reflux; (e) TFA/DCM; (f) R⁴-TH/K₂CO₃/DMF.

In scheme IV above, the following abbreviations are used: AcOH is aceticacid and R⁴-TH can be 2-chlorophenol, 3-chlorophenol, 3-fluorophenol or2,4-dichlorophenol or thiophenol or 3-fluorothiophenol.(4-oxo-4H-quinazolin-3-yl) acid derivatives A (X═N) are prepared inchiral form using methods analogous to Makino et al., Synlett, 11, 1670(2000). Commercially available 5-chloro-2-nitrobenzoic acid methyl esterJ is hydrolysed to the acid using lithium hydroxide and the resultingacid coupled to commercially available (S)-2-aminobutyric acidtert-butyl ester to give amide K. Reduction of the nitro group using tin(II) chloride followed by acid catalysed cyclo-condensation withtrimethylorthoformate furnishes the quinazoline, which is deprotectedwith trifluoroacetic acid to give the 4-oxo-4H-quinazolin-3-yl acid L.The acid is then coupled to the amino alcohol B (Scheme I).Alternatively, 5-chloro-2-nitrobenzoic acid methyl ester J can bereacted with a suitably substituted phenol or thiophenol by heating withpotassium carbonate in DMF to give the corresponding 6-phenoxy (Y═O) or6-phenylsulfanyl (Y═S) derivative. Treatment with lithium hydroxidegives the acid M, which can be further elaborated (Scheme IV, steps b-e)to give 4-oxo-4H-quinazolin-3-yl acid N which is then coupled to aminoalcohol B as previously described. 2-Nitrobenzoic acid may also beelaborated in an analogous manner to furnish the unsubstituted4-oxo-4H-quinazolin-3-yl acid.

Accordingly one aspect of this invention relates to a general method ofpreparing a compound of formula I, comprising the steps of:

reacting an acid or acid derivative of formula II,

-   -   with an amino alcohol of formula B to provide a compound of        formula III,

converting intermediate III to compound I, wherein; R⁷ is a suitableprotecting group, and Y, X, R², R³, and R⁴ are as described in any ofthe embodiments herein. A suitable protecting group would be known toskilled practitioners (see e.g., “Protective Groups in OrganicSynthesis” Third Ed. Greene, T. W. and Wuts, P. G., Eds., John Wiley &Sons, New York: 1999, the entire contents of which is herebyincorporated by reference). Preferably R⁷ is a C₁₋₆ straight or branchedalkyl. More preferably, R⁷ is t-butyl.

The compound of formula II and the compound of formula B may be reactedunder any conditions for coupling an amino compound and an acidcompound. Such conditions are well known to skilled practitioners.Preferred coupling conditions are those described in the schemes andexamples herein.

The compound of formula III may be converted to a compound of formula Iunder any conditions for converting a hydroxy group to a carbonyl groupand converting a protected acid to the free acid. Such conditions arewell known to skilled practitioners. Preferred conditions for preparinga carbonyl group from a hydroxyl group are those oxidizing conditionsdescribed in the schemes and examples herein. Preferred conditions fordeprotecting a protected acid when R⁷ is t-butyl are those hydrolysisconditions described in the schemes and examples herein.

This method is particularly useful for preparing chiral compounds ofthis invention, where the carbon bearing the R² substituent isstereochemically enriched. As exemplified below (see e.g., Examples1-22), intermediate acids or acid derivatives of formula II may beobtained in chiral form. This is illustrated herein forquinazolin-4-ones where X is nitrogen (see e.g., Examples 5-8, 12-18,20, and 22) and for isoquinolin-1-ones where X is CH (see e.g., Examples1-4, 9-11, 19, and 21). The coupling of II and B to provide III may becarried out according to any suitable method. The conversion of III toprovide I may be performed as described herein or according to othermethods familiar to those skilled in the art.

Certain chiral intermediates of II are useful in processes for preparingcompounds of this invention. A preferred intermediate is represented bycompound IIA:

wherein X, R², R³, and R⁴ are as described in any of the embodimentsherein.

According to a more preferred embodiment for compounds of formula II andIIA, R² is ethyl or isopropyl.

The compounds of this invention can be assayed for their ability toinhibit apoptosis, the release of IL-1β or caspase activity directly.Assays for each of the activities are known in the art and are describedbelow in detail in Examples 23-25.

If pharmaceutically acceptable salts of the compounds of this inventionare utilized in these compositions, those salts are preferably derivedfrom inorganic or organic acids and bases. Included among such acidsalts are the following: acetate, adipate, alginate, aspartate,benzoate, benzene sulfonate, bisulfate, butyrate, citrate, camphorate,camphor sulfonate, cyclopentanepropionate, digluconate, dodecylsulfate,ethanesulfonate, fumarate, glucoheptanoate, glycerophosphate,hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide,hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate,methanesulfonate, 2-naphthalenesulfonate, nicotinate, oxalate, pamoate,pectinate, persulfate, 3-phenyl-propionate, picrate, pivalate,propionate, succinate, tartrate, thiocyanate, tosylate and undecanoate.Base salts include ammonium salts, alkali metal salts, such as sodiumand potassium salts, alkaline earth metal salts, such as calcium andmagnesium salts, salts with organic bases, such as dicyclohexylaminesalts, N-methyl-D-glucamine, and salts with amino acids such asarginine, lysine, and so forth.

Also, the basic nitrogen-containing groups can be quaternized with suchagents as lower alkyl halides, such as methyl, ethyl, propyl, and butylchloride, bromides and iodides; dialkyl sulfates, such as dimethyl,diethyl, dibutyl and diamyl sulfates, long chain halides such as decyl,lauryl, myristyl and stearyl chlorides, bromides and iodides, aralkylhalides, such as benzyl and phenethyl bromides and others. Water oroil-soluble or dispersible products are thereby obtained.

The compounds utilized in the compositions and methods of this inventionmay also be modified by appending appropriate functionalities to enhanceselective biological properties. Such modifications are known in the artand include those which increase biological penetration into a givenbiological system (e.g., blood, lymphatic system, central nervoussystem), increase oral availability, increase solubility to allowadministration by injection, alter metabolism and alter rate ofexcretion.

Pharmaceutically acceptable carriers that may be used in thesecompositions include, but are not limited to, ion exchangers, alumina,aluminum stearate, lecithin, serum proteins, such as human serumalbumin, buffer substances such as phosphates, glycine, sorbic acid,potassium sorbate, partial glyceride mixtures of saturated vegetablefatty acids, water, salts or electrolytes, such as protamine sulfate,disodium hydrogen phosphate, potassium hydrogen phosphate, sodiumchloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, cellulose-based substances, polyethylene glycol, sodiumcarboxymethylcellulose, polyacrylates, waxes,polyethylene-polyoxypropylene-block polymers, polyethylene glycol andwool fat.

According to a preferred embodiment, the compositions of this inventionare formulated for pharmaceutical administration to a mammal, preferablya human being.

Such pharmaceutical compositions of the present invention may beadministered orally, parenterally, by inhalation spray, topically,rectally, nasally, buccally, vaginally or via an implanted reservoir.The term “parenteral” as used herein includes subcutaneous, intravenous,intramuscular, intra-articular, intra-synovial, intrasternal,intrathecal, intrahepatic, intralesional and intracranial injection orinfusion techniques. Preferably, the compositions are administeredorally or intravenously.

Sterile injectable forms of the compositions of this invention may beaqueous or oleaginous suspension. These suspensions may be formulatedaccording to techniques known in the art using suitable dispersing orwetting agents and suspending agents. The sterile injectable preparationmay also be a sterile injectable solution or suspension in a non-toxicparenterally-acceptable diluent or solvent, for example as a solution in1,3-butanediol. Among the acceptable vehicles and solvents that may beemployed are water, Ringer's solution and isotonic sodium chloridesolution. In addition, sterile, fixed oils are conventionally employedas a solvent or suspending medium. For this purpose, any bland fixed oilmay be employed including synthetic mono- or di-glycerides. Fatty acids,such as oleic acid and its glyceride derivatives are useful in thepreparation of injectables, as are natural pharmaceutically-acceptableoils, such as olive oil or castor oil, especially in theirpolyoxyethylated versions. These oil solutions or suspensions may alsocontain a long-chain alcohol diluent or dispersant, such ascarboxymethyl cellulose or similar dispersing agents, which arecommonly, used in the formulation of pharmaceutically acceptable dosageforms including emulsions and suspensions. Other commonly usedsurfactants, such as Tweens, Spans and other emulsifying agents orbioavailability enhancers which are commonly used in the manufacture ofpharmaceutically acceptable solid, liquid, or other dosage forms mayalso be used for the purposes of formulation.

The pharmaceutical compositions of this invention may be orallyadministered in any orally acceptable dosage form including, but notlimited to, capsules, tablets, aqueous suspensions or solutions. In thecase of tablets for oral use, carriers, which are commonly used, includelactose and corn starch. Lubricating agents, such as magnesium stearate,are also typically added. For oral administration in a capsule form,useful diluents include lactose and dried corn starch. When aqueoussuspensions are required for oral use, the active ingredient is combinedwith emulsifying and suspending agents. If desired, certain sweetening,flavoring or coloring agents may also be added.

Alternatively, the pharmaceutical compositions of this invention may beadministered in the form of suppositories for rectal administration.These can be prepared by mixing the agent with a suitable non-irritatingexcipient, which is solid at room temperature but liquid at rectaltemperature and therefore will melt in the rectum to release the drug.Such materials include cocoa butter, beeswax and polyethylene glycols.

The pharmaceutical compositions of this invention may also beadministered topically, especially when the target of treatment includesareas or organs readily accessible by topical application, includingdiseases of the eye, the skin, or the lower intestinal tract. Suitabletopical formulations are readily prepared for each of these areas ororgans.

Topical application for the lower intestinal tract can be effected in arectal suppository formulation (see above) or in a suitable enemaformulation. Topically-transdermal patches may also be used.

For topical applications, the pharmaceutical compositions may beformulated in a suitable ointment containing the active componentsuspended or dissolved in one or more carriers. Carriers for topicaladministration of the compounds of this invention include, but are notlimited to, mineral oil, liquid petrolatum, white petrolatum, propyleneglycol, polyoxyethylene, polyoxypropylene compound, emulsifying wax andwater. Alternatively, the pharmaceutical compositions can be formulatedin a suitable lotion or cream containing the active components suspendedor dissolved in one or more pharmaceutically acceptable carriers.Suitable carriers include, but are not limited to, mineral oil, sorbitanmonostearate, polysorbate 60, cetyl esters wax, cetearyl alcohol,2-octyldodecanol, benzyl alcohol and water.

For ophthalmic use, the pharmaceutical compositions may be formulated asmicronized suspensions in isotonic, pH adjusted sterile saline, or,preferably, as solutions in isotonic, pH adjusted sterile saline, eitherwith our without a preservative such as benzylalkonium chloride.Alternatively, for ophthalmic uses, the pharmaceutical compositions maybe formulated in an ointment such as petrolatum.

The pharmaceutical compositions of this invention may also beadministered by nasal aerosol or inhalation. Such compositions areprepared according to techniques well-known in the art of pharmaceuticalformulation and may be prepared as solutions in saline, employing benzylalcohol or other suitable preservatives, absorption promoters to enhancebioavailability, fluorocarbons, and/or other conventional solubilizingor dispersing agents.

The above-described compositions are particularly useful in therapeuticapplications relating to an IL-1 mediated disease, an apoptosis mediateddisease, an inflammatory disease, an autoimmune disease, a destructivebone disorder, a proliferative disorder, an infectious disease, adegenerative disease, a disease associated with cell death, an excessdietary alcohol intake disease, a viral mediated disease, retinaldisorders, uveitis, inflammatory peritonitis, osteoarthritis,pancreatitis, asthma, adult respiratory distress syndrome,glomerulonephritis, rheumatoid arthritis, systemic lupus erythematosus,scleroderma, chronic thyroiditis, Grave's disease, autoimmune gastritis,diabetes, autoimmune hemolytic anemia, autoimmune neutropenia,thrombocytopenia, chronic active hepatitis, myasthenia gravis,inflammatory bowel disease, Crohn's disease, psoriasis, atopicdermatitis, scarring, graft vs host disease, organ transplant rejection,organ apoptosis after burn injury, osteoporosis, leukemia's and relateddisorders, myelodysplastic syndrome, multiple myeloma-related bonedisorder, acute myelogenous leukemia, chronic myelogenous leukemia,metastatic melanoma, Kaposi's sarcoma, multiple myeloma, haemorrhagicshock, sepsis, septic shock, burns, Shigellosis, Alzheimer's disease,Parkinson's disease, Huntington's disease, Kennedy's disease, priondisease, cerebral ischemia, epilepsy, myocardial ischemia, acute andchronic heart disease, myocardial infarction, congestive heart failure,atherosclerosis, coronary artery bypass graft, spinal muscular atrophy,amyotrophic lateral sclerosis, multiple sclerosis, HIV-relatedencephalitis, aging, alopecia, neurological damage due to stroke,ulcerative colitis, traumatic brain injury, spinal cord injury,hepatitis-B, hepatitis-C, hepatitis-G, yellow fever, dengue fever, orJapanese encephalitis, various forms of liver disease, renal disease,polycystic kidney disease, H. pylori-associated gastric and duodenalulcer disease, HIV infection, tuberculosis, an immunotherapy for thetreatment of various forms of cancer, organ failure, and meningitis. Thecompounds and compositions are also useful in treating complicationsassociated with coronary artery bypass grafts. The amount of compoundpresent in the above-described compositions should be sufficient tocause a detectable decrease in the severity of the disease or in caspaseactivity and/or cell apoptosis, as measured by any of the assays knownin the art.

According to another embodiment, the compositions of this invention mayfurther comprise another therapeutic agent. Such agents include, but arenot limited to, thrombolytic agents such as tissue plasminogen activatorand streptokinase. When a second agent is used, the second agent may beadministered either as a separate dosage form or as part of a singledosage form with the compounds or compositions of this invention. Boththe compound and the additional agent should be present at dosage levelsof between 10 to 100%, and more preferably between about 10 to 80% ofthe dosage normally administered in a monotherapy regimen.

It should also be understood that a specific dosage and treatmentregimen for any particular patient will depend upon a variety offactors, including the activity of the specific compound employed, theage, body weight, general health, sex, diet, time of administration,rate of excretion, drug combination, and the judgment of the treatingphysician and the severity of the particular disease being treated. Theamount of active ingredients will also depend upon the particularcompound and other therapeutic agent, if present, in the composition.

In certain embodiments, the compounds of the invention may beadministered orally or parenterally at dosage levels of about 0.01 mg/kgto about 50 mg/kg and preferably from about 1 mg/kg to about 25 mg/kg,of subject body weight per day, one or more times a day, to obtain thedesired therapeutic effect.

In a preferred embodiment, the invention provides a method of treating amammal, having one of the aforementioned diseases, comprising the stepof administering to said mammal a pharmaceutically acceptablecomposition described above. In this embodiment, if the patient is alsoadministered another therapeutic agent or caspase inhibitor, it may bedelivered together with the compound of this invention in a singledosage form, or, as a separate dosage form. When administered as aseparate dosage form, the other caspase inhibitor or agent may beadministered prior to, at the same time as, or following administrationof a pharmaceutically acceptable composition comprising a compound ofthis invention.

The compounds of this invention or pharmaceutically acceptablecompositions thereof may also be incorporated into compositions forcoating implantable medical devices, such as prostheses, artificialvalves, vascular grafts, stents and catheters. Accordingly, the presentinvention, in another aspect, includes a composition for coating animplantable device comprising a compound of the present invention asdescribed generally above, and in classes and subclasses herein, and acarrier suitable for coating said implantable device. In still anotheraspect, the present invention includes an implantable device coated witha composition comprising a compound of the present invention asdescribed generally above, and in classes and subclasses herein, and acarrier suitable for coating said implantable device.

Another aspect of the invention relates to inhibiting caspase activityin a biological sample or a patient, which method comprisesadministering to the patient, or contacting said biological sample witha compound of formula I or a composition comprising said compound. Theterm “biological sample”, as used herein, includes, without limitation,cell cultures or extracts thereof; biopsied material obtained from amammal or extracts thereof; and blood, saliva, urine, feces, semen,tears, or other body fluids or extracts thereof.

Inhibition of caspase activity in a biological sample is useful for avariety of purposes that are known to one of skill in the art. Examplesof such purposes include, but are not limited to, blood transfusion,organ-transplantation, biological specimen storage, and biologicalassays.

The compounds of this invention are useful in methods for preservingcells, such as may be needed for an organ transplant or for preservingblood products. Similar uses for caspase inhibitors have been reported(Schierle et al., Nature Medicine, 5, 97 (1999)). The method involvestreating the cells or tissue to be preserved with a solution comprisingthe caspase inhibitor. The amount of caspase inhibitor needed willdepend on the effectiveness of the inhibitor for the given cell type andthe length of time required to preserve the cells from apoptotic celldeath.

In order that this invention be more fully understood, the followingpreparative and testing examples are set forth. These examples are forthe purpose of illustration only and are not to be construed as limitingthe scope of the invention in any way.

Example 1(S)-3-[2-(7-chloro-1-oxo-1H-isoquinolin-2-yl)-butyrylamino]-5-fluoro-4-oxo-pentanoicacid

Method A: 5-Chloro-2(2-methoxyvinyl)-benzoic acid

To a cooled (0° C.) slurry of methoxymethyltriphenylphosphonium chloride(39 g) in a mixture of diethyl ether (200 ml) and tert-butanol (50 ml)was added potassium tert-butoxide (12.8 g) portion wise. The resultingmixture was stirred at 0° C. for 1 hour, then a solution of2-formyl-5-chlorobenzoic acid (prepared as described in J. Org. Chem.61, 3402 (1996)) (10 g) in diethyl ether (50 ml) was added dropwise over15 minutes. The resulting mixture was stirred for 1 hour at 0° C., thenwarmed to ambient and stirred for an additional 90 minutes. The mixturewas diluted with water (200 ml) and the organic phase removed. Theaqueous phase was acidified to pH 1 with 1M HCl and extracted with ethylacetate (3×50 ml). The combined extracts were washed with brine, dried(magnesium sulfate), filtered and concentrated. The residue was purifiedby flash chromatography (50% ethyl acetate/hexane) to afford thesub-title compound as a yellow solid (9.13 g, 80%). ¹H NMR (400 MHz,CDCl₃) δ 3.70-3.81 (3H, s), 6.20 (0.3H, d, cis alkene), 6.30 (0.3H, d,cis alkene), 6.80 (0.7H, d, trans alkene), 7.01 (0.7H, d, trans alkene),7.30-8.15 (3H, m) ppm.

Method B: 7-Chloro-isochromen-1-one

Concentrated sulphuric acid (15 ml) was added to5-chloro-2(2-methoxyvinyl)-benzoic acid (4.43 g) at 0° C. The mixturewas stirred for 2 hours, then diluted with ice/water. The product wasextracted with ethyl acetate (3×15 ml) and the combined extracts washedwith saturated sodium bicarbonate solution. The solution was dried(magnesium sulfate), filtered and concentrated. The residue was purifiedby flash chromatography (0-5% ethyl acetate/hexane) to afford thesub-title compound as a white solid (3.04 g, 81%). mp 109.8-110.9° C. ¹HNMR (400 MHz, CDCl₃) δ 6.51 (1H, d), 7.28-7.32 (1H, m), 7.41 (1H, d),7.64-7.70 (1H, m), 8.28 (1H, m) ppm.

Method C:(S)-2-[3-(1-tertButoxycarbonyl-propylamino)-7-chloro-1-oxo-3,4-dihydro-1H-isoquinolin-2-yl]-butyricacid tert-butyl ester

A mixture of 7-chloro-isochromen-1-one (10 g) and (S)-2-aminobutyricacid, tert butyl ester (22 g) were heated at 85° C. for 24 hours. Themixture was then cooled and purified by flash chromatography (5-25%ethyl acetate/hexane) to afford the sub-title compound as a yellow oil(17.1 g, 64%). ¹H NMR (400 MHz, CDCl₃) δ 0.68-1.32 (6H, m), 1.50 (21H,m), 1.92 (1H, m), 2.15 (1H, m), 2.82-3.40 (3H, m), 4.41 (1H, m), 4.68(1H, m), 7.11 (1H, m), 7.35-7.52 (1H, m), 8.05 (1H, m) ppm.

Method D: (S)-2-(7-Chloro-1-oxo-1H-isoquinolin-2-yl)-butyric acid, tertbutyl ester

To a stirred solution of2-[3-(1-tertbutoxycarbonyl-propylamino)-7-chloro-1-oxo-3,4-dihydro-1H-isoquinolin-2-yl]-butyricacid tert-butyl ester (8.58 g) in isopropanol (180 ml) at 0° C. wasadded concentrated hydrochloric acid (20 ml). The resulting mixture wasallowed to warm to room temperature and stirred for 18 hours. Themixture was then diluted with ethyl acetate (500 ml) and water (150 ml).The organic phase was separated and washed with water, then brine, dried(magnesium sulfate), filtered and concentrated. The sub-title productwas obtained as a yellow solid (5.57 g, 97%). m.p. 111.3-111.8° C.;[α]²⁵ _(D) −52.3° (c=1, CHCl₃); IR (solid) 1731.4, 1649.5, 1593.2,1229.6, 1152.8, 901.9 cm⁻¹; ¹H NMR (400 MHz, CDCl₃) δ 0.95 (3H, t), 1.48(9H, s), 1.95 (1H, m), 2.30 (1H, m), 5.55 (1H, m), 6.40 (1H, m), 7.15(1H, m), 7.49 (1H, m), 7.61 (1H, m), 8.40 (1H, m) ppm; ¹³C NMR (100 MHz,CDCl₃) δ 10.9, 24,8, 28.1, 59.2, 82,8, 105.7, 127.3, 127.8, 128.1,129.5, 133.1, 133.2, 135.4, 161.8, 170.2; MS ES(+) 322.4 (M+H).

Method E: (S)-2-(7-Chloro-1-oxo-1H-isoquinolin-2-yl)-butyric acid

A solution of (S)-2-(7-chloro-1-oxo-1H-isoquinolin-2-yl)-butyric acid,tert-butyl ester (322 mg) in dichloromethane (14 ml) was cooled to 0° C.Trifluoroacetic acid (3.5 ml) was added and the resulting mixtureallowed to warm to room temperature and stirred for 2 hours. The mixturewas then concentrated under reduced pressure and the residuere-dissolved in dichloromethane. This process was repeated several timesin order to remove excess trifluoroacetic acid. The resulting solid wasslurried in diethyl ether, filtered and washed with more diethyl ether.The solid was then dried to constant weight under vacuum. This gave thesub-title product as a white solid (236 mg, 89%). m.p. 159.6-160.1° C.;[α]²⁴ _(D) −47.0° (c=1.01, CHCl₃); IR (solid) 1731.4, 1639.3, 1577.8,1209.1, 1168.1 cm⁻¹; ¹H NMR (400 MHz, d₆-DMSO) δ 0.82 (3H, t), 2.00-2.25(2H, m), 5.20 (1H, m), 6.70 (1H, d), 7.49 (1H, d), 7.70-7.81 (2H, m),8.18 (1H, s) ppm; ¹³C NMR (100 MHz, d₆-DMSO) δ 10.8, 22.7, 60.8, 104.9,126.5, 126.6, 128.8, 131.6, 132.5, 133.1, 135.8, 160.5, 171.7; MS ES (+)266.27 (M+H).

Method F:(S)-3-[2-(7-Chloro-1-oxo-1H-isoquin-2-yl)-butyrylamino]-5-fluoro-4-hydroxy-pentanoicacid tert-butyl ester

A stirred mixture of (S)-2-(7-chloro-1-oxo-1H-isoquinolin-2-yl)-butyricacid (15 g), 3-amino-5-fluoro-4-hydroxy-pentanoic acid tert-butyl ester(prepared as described in Tetrahedron Lett. 35, 9693 (1994)) (12.9 g),HOBt (8.4 g), DMAP (7.2 g) and THF (450 ml) was cooled to 0° C. then EDC(11.9 g) was added. The mixture was allowed to warm to room temperatureduring 16 h then concentrated under reduced pressure. The residue waspurified by flash chromatography (30-60% ethyl acetate/hexane) to affordthe subtitle compound as a white foam (24.6 g, 96%). ¹H NMR (400 MHz,CDCl₃) δ 0.92 (3H, m), 1.13-1.50 (9H, m), 1.95 (1H, m), 2.25 (1H, m),2.45-2.78 (2H, m), 3.68-4.60 (5H, m), 5.50 (1H, m), 6.60 (1H, m),7.21-7.60 (4H, m), 8.20-8.31 (1H, m) ppm; ¹⁹F NMR (376 MHz, CDCl₃)(proton decoupled) δ −229.6, −229.7, −230.5, −230.6.

Method G:(S)-3-[2-(7-Chloro-1-oxo-1H-isoquin-2-yl)-butyrylamino]-5-fluoro-4-oxo-pentanoicacid tert-butyl ester

A stirred solution of3-[2-(7-chloro-1-oxo-1H-isoquin-2-yl)-butyrylamino]-5-fluoro-4-hydroxy-pentanoicacid tert-butyl ester (47.8 g) in anhydrous DCM (1.2 L) was treated with1,1,1-triacetoxy-1,1-dihydro-1,2-benziodoxol-3(1H)-one (53.5 g) at 0° C.The resulting mixture was kept at 0° C. for 2 hrs., diluted with ethylacetate, then poured into a 1:1 mixture of saturated aqueous sodiumhydrogen carbonate and saturated aqueous sodium thiosulfate. The organiclayer was removed and the aqueous layer re-extracted with ethyl acetate.The combined organic extracts were dried (magnesium sulfate), filteredand concentrated. The residue was purified by flash chromatography(20-40% ethyl acetate/hexane) to afford the subtitle compound as a whitesolid (41.9 g, 88%). ¹H NMR (400 MHz, CDCl₃) δ 1.00 (3H, t), 1.29 (5H,s), 1.41 (4H, s), 2.01 (1H, m), 2.29 (1H, m), 2.61-3.05 (2H, m), 4.77(3H, m), 5.50 (1H, m), 6.60 (1H, m), 7.20-7.34 (2H, m), 7.51 (1H, m),7.62 (1H, m), 8.41 (1H, m) ppm; ¹⁹F NMR (376 MHz, CDCl₃) (protondecoupled) δ −231.89, −232.30.

(S)-3-[2-(7-chloro-1-oxo-1H-isoquinolin-2-yl)-butyrylamino]-5-fluoro-4-oxo-pentanoicacid

This was prepared using a procedure similar to that described in methodE. The product was isolated as a white solid (98%). IR (solid) 1782.7,1741.7, 1644.4, 1593.2, 1536.8, 1209.1, 1168.1, 1055.5, 840.4 cm⁻¹; ¹HNMR (400 MHz, d₆-DMSO) δ 0.82 (3H, m), 1.81-2.25 (2H, m), 2.25-3.11 (2H,m), 4.15-5.60 (4H, m), 6.70 (1H, m), 7.55 (1H, m), 7.78 (2H, m), 8.15(1H, s), 8.35-9.00 (1H, brm) ppm; ¹³C NMR (100 MHz, d₆-DMSO) δ 10.6,23.0, 24.0, 24.6, 32.9, 34.6, 34.7, 47.7, 52.2, 52.3, 58.2, 58.23, 58.7,59.1, 83.4, 83.5, 85.2, 85.3, 103.9, 104.5, 104.7, 104.8, 126.5, 126.6,128.8, 131.3, 131.4, 131., 133.1, 135.7, 135.73, 160.8, 170.2, 170.3,170.4, 172.0, 173.1, 202.6, 202.7; ¹⁹F NMR (376 MHz, d₆-DMSO) (protondecoupled) δ −226.70, −226.75, −227.51, −230.5, −231.16, −232.61,−232.67, −233.37; MS ES (−) 395.33 (M−H).

Example 2(S)-3-[2-(7-trifluoromethyl-1-oxo-1H-isoquinolin-2-yl)-butyrylamino]-5-fluoro-4-oxo-pentanoicacid

This was prepared using procedures similar to those described in methodsA-G (2-formyl-5-trifluoromethylbenzoic acid was prepared using aprocedure similar to that described in J. Org. Chem. 61, 3402 (1996).The product was isolated as a white solid (93% last step). IR (solid)1782.6, 1746.8, 1644.4, 1629.0, 1603.4, 1321.8, 1275.7, 1168.1, 1127.2,927.5 cm⁻¹; ¹H NMR (400 MHz, d₆-DMSO) δ 0.82 (3H, m), 1.85-2.21 (2H, m),2.35-3.21 (2H, m), 4.20-5.75 (4H, m), 6.80 (1H, m), 7.68 (1H, m), 7.92(1H, m), 8.04 (1H, m), 8.49 (1H, s), 8.56-8.95 (1H, brm) ppm; ¹⁹F NMR(376 MHz, d₆-DMSO) (proton decoupled) δ −61.32, −61.38, −226.70,−226.76, −230.47, −231.06, −232.61, −232.67; MS ES (−) 429.30 (M−H).

Example 3(S)-3-[2-(6-chloro-1-oxo-1H-isoquinolin-2-yl)-butyrylamino]-5-fluoro-4-oxo-pentanoicacid

Method H: 4-Chloro-N-methyl-benzamide

To a 0° C. solution of the 4-chlorobenzoyl chloride (4.50 g) indichloromethane (10 mL) was added an 8M solution of methylamine inethanol dropwise. The solution was stirred for 16 h and then evaporatedto dryness. The residue was diluted with saturated sodium bicarbonatesolution (10 mL) and extracted three times with ethyl acetate (3×20 mL),the organics washed with brine (10 mL), dried (MgSO₄) and concentratedin vacuo to afford the sub-title compound as a white solid (4.33 g;97%). ¹H NMR (400 MHz, CDCl₃) δ 3.00 (3H, s), 7.40 (1H, br) 7.40 (1H,d), 7.70 (1H, d) ppm.

Method I: 2-formyl-4-chloro-N-methylbenzamide

To a solution of 4-chloro-N-methyl-benzamide (3.1 g) in THF (30 mL) wasadded n-butyl lithium (30.1 mL of 2.5M hexane solution) and the solutionrefluxed for 45 min. The solution was then cooled to 0° C. andN-methylformanilide (9.27 mL) added dropwise over 2 min. The solutionwas then refluxed for 2 h, then cooled to ambient temperature, water (80mL) added and the solution acidified to pH 1 with 2M HCl. The solutionwas then extracted three times with ethyl acetate (3×50 mL), washed withbrine (20 mL), dried (MgSO₄) and concentrated in vacuo. The resultingbrown oil was purified on silica by flash chromatography to afford thesub-titled product as a pale yellow solid (2.13 g; 59%). ¹H NMR (400MHz, CDCl₃) δ 2.90 (3H, s), 4.25 (1H, d, J) 5.60 (1H, d, J), 7.35 (2H,s), 7.60 (1H, s) ppm.

Method J 2-Formyl-4-chlorobenzoic acid

A mixture of 2-formyl-4-chloro-N-methylbenzamide (3.19 g) and 10Mhydrochloric acid (30 ml) was heated at reflux for 18 hours. The mixturewas cooled and basified with saturated sodium hydrogen carbonatesolution. The solution was then washed with ethyl acetate, thenacidified with 2M hydrochloric acid. The product was extracted withethyl acetate and the combined extracts dried with magnesium sulfate.The solution was then filtered and concentrated. This furnished2-formyl-4-chlorobenzoic acid as a yellow solid (2.22 g, 75%). ¹H NMR(400 MHz, CDCl₃) δ 6.65 (0.5H, brs), 7.50 (2H, m), 7.65 (1H, m), 7.85(0.5H, brm), 8.05 (1H, m) ppm.

(S)-3-[2-(6-chloro-1-oxo-1H-isoquinolin-2-yl)-butyrylamino]-5-fluoro-4-oxo-pentanoicacid

This was prepared from 2-formyl-4-chlorobenzoic acid (prepared asdescribed in methods H-J) using procedures similar to those described inmethods A-G. The title compound was isolated by preparative HPLC and wasobtained as a white solid. ¹H NMR (400 MHz, CDCl₃) δ 0.97 (3H, m),1.90-2.31 (2H, m), 2.65-3.30 (2H, m), 4.20-5.75 (4H, m), 6.65 (1H, m),7.40-7.60 (3H, m), 8.29 (1H, m), 9.20 (1H, br) ppm; ¹⁹F NMR (376 MHz,CDCl₃) (proton decoupled) δ −229.80, −232.07, −232.43, −232.58, −232.78;MS ES (−) 395.26 (M−H).

Example 4(S)-3-[2-(6-trifluoromethyl-1-oxo-1H-isoquinolin-2-yl)-butyrylamino]-5-fluoro-4-oxo-pentanoicacid

This was prepared from 2-formyl-4-trifluoromethylbenzoic acid (preparedfrom 4-trifluoromethylbenzoic acid using methods similar to thosedescribed in H-J) using procedures similar to those described in methodsA-G. The title compound was isolated as a white solid (95%, last step).¹H NMR (400 MHz, CDCl₃) δ 0.99 (3H, m), 1.90-2.30 (2H, m), 2.60-3.50(2H, m), 4.20-5.75 (4H, m), 6.80 (1H, m), 7.50-7.90 (3H, m), 7.92 (1H,m), 8.40-8.60 (1H, m) ppm; ¹⁹F NMR (376 MHz, d₆-DMSO) (proton decoupled)δ −63.60, −63.61, −63.65, −231.67, −231.80, −232.06, −232.18; MS ES(+)431.26 (M+H).

Example 5(S,S)-3-[2-(6-Chloro-4-oxo-4H-quinazolin-3-yl)-butyrylamino]-4-oxo-5-(2,3,5,6-tetrafluoro-phenoxy)pentanoicacid

Method K: (S)-2-(5-chloro-2-nitro-benzoylamino)-butyric acid-tert-butylester

To a stirred solution of 5-chloro-2-nitro-benzoic acid (4.68 g) inanhydrous tetrahydrofuran (100 ml) at 0° C., was added(S)-2-amino-butyric acid-tert-butyl ester hydrochloric acid salt (5 g),HOBt (3.45 g), DMAP (2.98 g), followed by EDC (4.88 g) anddiisopropylethylamine (3.30 g). The reaction mixture was stirred for 10minutes then warmed to room temperature and stirred for 16 hours beforeconcentration at reduced pressure. The resulting residue was partitionedbetween EtOAc (100 ml) and saturated sodium bicarbonate solution (100ml), the organic layer separated and further washed with 1M hydrochloricacid and saturated brine solution, dried (MgSO4), filtered andconcentrated to give the sub-title compound as a pale yellow oil (6.82g, 68% yield). ¹H NMR (400 MHz, CDCl₃) δ 8.05 (1H, d), 7.55 (2H, m), 6.4(1H, m) 4.7 (1H, m), 2.1 (1H, m), 1.95 (1H, m), 1.5 (9H, s), 1.0 (3H, t)ppm.

Method L: (S)-2-(2-amino-5-chloro-benzoylamino)-butyric acid-tert-butylester

To a stirred solution of (S)-2-(5-chloro-2-nitro-benzoylamino)-butyricacid-tert-butyl ester (5.72 g,) in ethanol (250 ml) at ambienttemperature was added tin(II) chloride bis-hydrate (18.8 g). The mixturewas stirred at ambient for 16 hours. The volume was reduced toapproximately 50 ml under reduced pressure, and reaction mixture wasthen diluted with 1M NaOH until the precipitate formed re-dissolved (400ml). The aqueous was then extracted with diethyl ether (3×100 ml). Theorganic extracts were combined and washed with brine, dried (MgSO₄), andconcentrated to give the sub-titled compound as a yellow solid (4.60 g,88% yield). ¹H NMR (400 MHz, CDCl₃) δ 7.4 (1H, s), 7.2 (1H, d), 6.6 (2H,m), 5.5 (2H, m), 4.6 (1H, m), 2.0 (1H, m), 1.85 (1H, m), 1.5 (9H, s),0.95 (3H, t) ppm.

Method M: (S)-2-(6-chloro-4-oxo-4H-quinazoline-3-yl)-butyricacid-tert-butyl ester

2-(2-amino-5-chloro-benzoylamino)-butyric acid-tert-butyl ester (2.82 g)was dissolved in trimethylorthoformate (50 ml). Acetic acid (10 ml) wasadded, and reaction mixture refluxed for 48 hours. After this time, thesolvents were removed and the residue partitioned between ethyl acetateand saturated sodium bicarbonate solution. The aqueous layer was furtherextracted with ethyl acetate and the combined organic extracts washedwith brine, dried (MgSO₄) and concentrated under reduced pressure togive a yellow oil. This was purified by flash chromatography (20% EtOAc:Petroleum) to give the sub-title compound as an oil (1.23 g, 42% yield).¹H NMR (400 MHz, CDCl₃) δ 8.25 (1H, s), 8.05 (1H, s), 7.7 (2H, m), 5.3(1H, m), 2.3 (1H, m), 2.0 (1H, m), 1.5 (9H, s), 1.0 (3H, t) ppm.

2-(6-chloro-4-oxo-4H-quinazoline-3-yl)-butyric acid

This was prepared using a procedure similar to that described in methodE. The sub-title compound was obtained as an off-white solid. ¹H NMR(400 MHz, MeOD) δ 8.3 (1H, s), 8.15 (1H, s), 7.8 (1H, d), 7.7 (1H, d),5.2 (1H, m), 2.4 (1H, m), 2.2 (1H, m), 0.95 (3H, t) ppm.

Method N:(S)-3-Benzyloxycarbonylamino-4-oxo-5-(2,3,5,6-tetrafluoro-phenoxy)-pentanoicacid tert-butyl ester

Potassium fluoride (2.8 g) was added portionwise to a stirred solutionof (S)-3-benzyloxycarbonylamino-5-bromo-4-oxo-pentanoic acid tert-butylester (18.6 g) and 2,3,5,6-tetrafluorophenol (9.3 g) in anhydrous DMF(250 mL) under nitrogen at room temperature. The mixture was thenstirred for 18 hours before being quenched with ethyl acetate and water.The organic layer was removed and washed with sodium bicarbonatesolution, dried (magnesium sulfate) and concentrated to give thesub-title product as an off-white solid (21.1 g, 96%). ¹H NMR (400 MHz,CDCl₃) δ 1.43 (9H, s), 2.76 (1H, dd), 3.06 (1H, dd), 4.67-4.71 (1H, m),5.12 (1 h, d), 5.22 (1H, d), 5.86 (1H, d), 7.35-7.38 (5H, m) ppm; ¹⁹FNMR (376 MHz, CDCl₃) (proton decoupled) δ −139.98, −140.00, −140.04,−140.06, −157.05, −157.07, −157.11, −157.13; MS ES (+) 486.23 (M+H).

Method O:(3S)-3-Benzyloxycarbonylamino-4-hydroxy-5-(2,3,5,6-tetrafluoro-phenoxy)-pentanoicacid tert-butyl ester

NaBH₄ (1.65 g) was added portionwise to a stirred solution of3-benzyloxycarbonylamino-4-oxo-5-(2,3,5,6-tetrafluoro-phenoxy)-pentanoicacid tert-butyl ester (21.1 g) in anhydrous THF (220 mL) at −20° C.under nitrogen. After stirring at this temperature for 3 hours, thereaction was quenched by the addition of saturated ammonium chloridesolution and diluted with DCM. The organic layer was removed and theaqueous layer re-extracted with DCM. The combined organic extracts werewashed with brine, dried (magnesium sulfate) and concentrated. Theresidue was purified by column chromatography (10%-20% ethylacetate/hexane). The sub-title compound was obtained as a white solid(14.6 g, 73%). ¹H NMR (400 MHz, CDCl₃) δ 1.45 (9H, s), 2.61-2.77 (2H,m), 3.16-3.36 (1H, 2×brd d), 4.12-4.22 (2H, m), 4.30-4.33 (1H, m),5.44-5.69 (1H, 2×d), 6.78-6.86 (1H, m), 7.35-7.36 (5H, m) ppm; ¹⁹F NMR(346 MHz, CDCl₃) (proton decoupled) δ −139.87, −139.89, −139.93,−139.95, −139.98, −157.02, −157.05, −157.06, −157.08, −157.09, −157.10,−157.12; ES (+) 488.27 (M+H).

Method P:(3S)-3-Amino-4-hydroxy-5-(2,3,5,6-tetrafluoro-phenoxy)-pentanoic acidtert-butyl ester

10% Pd on carbon (2.92 g) was added portionwise to a solution of3-benzyloxycarbonylamino-4-hydroxy-5-(2,3,5,6-tetrafluoro-phenoxy)-pentanoicacid tert-butyl ester (14.6 g) in anhydrous MeOH (350 mL) which had beendegassed under nitrogen (5×). The reaction was further degassed undernitrogen (3×) and hydrogen (5×) and stirred at room temperature for 20minutes. The palladium catalyst was removed by filtration and thefiltrate concentrated to give the sub-titled compound as a white solid(9.5 g, 90%). ¹H NMR (400 MHz, CDCl₃) δ 1.49 (9H, s), 2.35-2.43 (1H, m),5.67-5.64 (1H, m), 3.37-3.43 (1H, m), 3.77-3.87 (1H, m), 4.28-4.63 (2H,m), 6.77-6.86 (1H, m) ppm; ¹⁹F NMR (346 MHz, CDCl₃) (proton decoupled) δ−139.95, −139.97, −140.00, −140.03, −140.05, −140.08, −140.11, −140.13,−157.15, −157.18, −157.21, −157.23, −157.27, −157.29.

(S,S)-3-[2-(6-Chloro-4-oxo-4H-quinazolin-3-yl)-butyrylamino]-4-oxo-5-(2,3,5,6-tetrafluoro-phenoxy)pentanoicacid

This compound was prepared using procedures similar to those describedin methods F, G, and E. The title compound was obtained as a white solid(TFA salt) (94%, last step). IR (solid) 3298.7, 1741.4, 1691.4, 1660.3,1601.1, 1517.4, 1490.8, 1320.6, 1244.7, 1174.5, 1104.4, 939.2, 836.5,715.5, 666.8 and 656.0 cm⁻¹; ¹H NMR (400 MHz, DMSO-d₆) δ 9.05 (1H, m),8.4 (1H, s), 8.05-7.5 (4H, m), 5.45-5.15 (3H, m), 4.6 (1H, m), 2.25-2.0(2H, m) and 0.9-0.75 (3H, m) ppm; ¹⁹F NMR (376 MHz, DMSO-d₆) (protondecoupled) δ −140.57, −140.59, −140.63, −140.65, −141.02, −141.04,−141.08, −141.10, −156.75, −156.77, −156.81, −156.83, −156.94, −156.96,−157.00, −157.02; MS ES (+) 544.2 (M+H).

Example 6(S,S)-4-Oxo-3-[2-(4-oxo-4H-quinazolin-3-yl)-butyrylamino]-5-(2,3,5,6-tetrafluoro-phenoxy)pentanoicacid

This compound was prepared using 2-(4-oxo-4H-quinazoline-3-yl)-butyricacid (synthesized from 2-nitrobenzoic acid using procedures similar tothose described in methods K-M and E) and(3S)-3-amino-4-hydroxy-5-(2,3,5,6-tetrafluoro-phenoxy)-pentanoic acidtert-butyl ester (prepared as described in methods N-P) using proceduressimilar to those described in methods F, G and E. The title compound wasobtained as a white solid (TFA salt) (85%, last step). IR (solid)1792.8, 1726.3, 1669.9, 1521.4, 1485.6, 1183.5, 1142.5, 1091.3 and 932.5cm⁻¹; ¹H NMR (400 MHz, DMSO-d₆) δ 9.0-8.65 (1H, m), 8.35 (1H, s), 8.1(1H, m), 7.8 (1H, m), 7.65 (1H, m), 7.55 (2H, m), 5.4-4.3 (4H, m),2.8-2.5 (2H, m), 2.2-2.0 (2H, m) and 0.9-0.7 (3H, m) ppm; ¹⁹F NMR (376MHz, DMSO-d₆) (proton decoupled) δ −140.57, −140.60, −140.63, −140.66,−141.03, −141.05, −141.09, −141.11, −156.77, −156.80, −156.83, −156.86,−156.94, −156.97, −157.00, −157.03; MS ES (+) 510.26 (M+H).

Example 7(S,S)-4-Oxo-3-[2-(4-oxo-4H-quinazolin-3-yl)-butyrylamino]-5-(2,4,6-trifluoro-phenoxy)-pentanoicacid

This compound was prepared using 2-(4-oxo-4H-quinazoline-3-yl)-butyricacid (synthesized from 2-nitrobenzoic acid using procedures similar tothose described in methods K-M and E) and(3S)-3-amino-4-hydroxy-5-(2,4,6-trifluoro-phenoxy)-pentanoic acidtert-butyl ester (prepared from 2,4,6-trifluorophenol using proceduressimilar to those described in methods N-P) using procedures similar tothose described in methods F, G and E. The title compound was obtainedas a white solid (TFA salt) (71%, last step). IR (solid) 3308.9, 3075.4,2928.4, 1803.1, 1726.9, 1672.7, 1612.1, 1510.0, 1478.7, 1452.8, 1407.6,1231.3, 1199.3, 1121.0, 1040.4, 997.6, 933.1, 839.7, 772.9, 720.3 and698.4 cm⁻¹; ¹H NMR (400 MHz, DMSO-d₆) δ 0.84-0.86 (3H, m), 2.04-2.95(4H,), 4.05-5.48 (5H, 6), 7.13-7.21 (2H, m), 7.54-7.57 (1H, m),7.69-7.72 (1H, m), 7.84-7.88 (1H, m), 8.07-8.13 (1H, m), 8.38 (1H, s),8.60-8.97 (1H, 2×dd) ppm; ¹⁹F NMR (376 MHz, DMSO-d₆) (proton decoupled)δ −115.4, −115.6, −124.9, −125.0, −125.6; MS ES (+): 492.3 (M+H).

Example 8(S,S)-4-Oxo-3-[2-(4-oxo-4H-quinazolin-3-yl)-butyrylamino]-5-(2,5,6-trifluoro-phenoxy)-pentanoicacid

This compound was prepared using 2-(4-oxo-4H-quinazoline-3-yl)-butyricacid (synthesized from 2-nitrobenzoic acid using procedures similar tothose described in methods K-M and E) and(3S)-3-amino-4-hydroxy-5-(2,3,6-trifluoro-phenoxy)-pentanoic acidtert-butyl ester (prepared from 2,3,6-trifluorophenol using proceduressimilar to those described in methods N-P) using procedures similar tothose described in methods F, G and E. The title compound was obtainedas a white solid (TFA salt)(87%, last step). IR (solid) 3308.9, 3075.4,2928.4, 1803.1, 1726.9, 1672.7, 1612.1, 1510.0, 1478.7, 1452.8, 1407.6,1231.3, 1199.3, 1121.0, 1040.4, 997.6, 933.1, 839.7, 772.9, 720.3 and698.4 cm⁻¹; ¹H NMR (400 MHz, DMSO-d₆) δ 0.84-0.86 (3H, m,), 2.04-2.95(4H, 3 m), 4.05-5.48 (5H, 6×m), 7.13-7.21 (2H, m,), 7.54-7.57 (1H, m),7.69-7.72 (1H, m), 7.84-7.88 (1H, m), 8.07-8.13 (1H, m), 8.38 (1H, s),8.60-8.97 (1H, 2) ppm; ¹⁹F NMR (376 MHz, DMSO) (proton decoupled) δ−115.4, −115.6, −124.9, −125.0, −125.6; MS ES (+): 492.3 (M+H).

Example 9(S,S)-3-[2-(1-oxo-1H-isoquinolin-2-yl)-butyrylamino]-4-oxo-5-(2,3,5,6-tetrafluoro-phenoxy)-pentanoicacid

This compound was prepared using(S)-2-(1-oxo-1H-isoquinolin-2-yl)-butyric acid (prepared from2-formylbenzoic acid using procedures similar to those described inmethods A-E) and(3S)-3-amino-4-hydroxy-5-(2,3,5,6-tetrafluoro-phenoxy)-pentanoic acidtert-butyl ester (prepared as described in methods N-P) using proceduressimilar to those described in methods F, G and E. The title compound wasisolated by preparative HPLC. IR (solid) 2960.2, 1780.1, 1746.2, 1646.6,1619.0, 1589.1, 1517.4, 1490.3, 1427.8, 1260.3, 1206.2, 1176.3, 1098.9,938.4, 788.1, 748.3, 714.2, 692.5, 665.3 and 655.8 cm⁻¹; ¹H NMR (400MHz, DMSO-d₆) δ 0.78-0.83 (3H, m), 1.89-1.96 (1H, m), 2.08-2.13 (1H, m),2.50-2.78 (2H, m), 4.35-5.45 (4H, 3×m), 6.64-6.69 (1H, m), 7.74-7.73(5H, m), 8.18-8.20 (1H, m), 8.81 & 8.90 (1H, d) ppm; ¹⁹F NMR (376 MHz,DMSO) (proton decoupled) δ −141.01, −141.03, −141.07, −141.09, −156.80,−156.82, −156.86, −156.88; MS ES (+) 509.2 (M+H).

Example 10(S,S)-3-[2-(7-chloro-1-oxo-1H-isoquinolin-2-yl)-butyrylamino]-4-oxo-5-(2,3,5,6-tetrafluoro-phenoxy)-pentanoicacid

This compound was prepared using(S)-2-(7-chloro-1-oxo-1H-isoquinolin-2-yl)-butyric acid (prepared asdescribed in methods A-E) and(3S)-3-amino-4-hydroxy-5-(2,3,5,6-tetrafluoro-phenoxy)-pentanoic acidtert-butyl ester (prepared as described in methods N-P) using proceduressimilar to those described in methods F, G and E. The product wasisolated as a white solid (94% last step). IR (solid) 1639.3, 1618.8,1593.2, 1516.4, 1485.6, 1219.4, 1168.1, 1106.7, 932.6, 830.2 cm⁻¹; ¹HNMR (400 MHz, DMSO-d₆) δ 0.80 (3H, t), 1.94-2.12 (2H, m), 2.55-2.61 (1H,m), 2.74-2.80 (1H, m), 4.58-4.63 (1H, m), 5.12-5.76 (3H, m), 6.70 (1H,d), 7.51-7.78 (4H, m), 8.11-8.12 (1H, m), 8.60-8.95 (1H, 2×d) ppm; ¹³CNMR (100 MHz, d₆-DMSO) δ 23.8, 24.5, 32.9, 34.6, 47.8, 52.8, 55.2, 58.2,58.9, 74.4, 75.6, 100.1, 100.3, 100.5, 101.0, 101.2, 104.6, 126.5,136.6, 131.3, 131.4, 133.0, 135.6, 135.6, 139.0, 139.1, 141.4, 141.6,144.6, 144.8, 144.9, 147.1, 147.1, 160.7, 170.4, 172.0, 173.0, 202.2;¹⁹F NMR (376 MHz, d₆-DMSO) (proton decoupled) δ −140.57, −140.60,−140.64, −140.66, −141.00, −141.03, −141.06, −141.09, −156.78, −156.80,−156.84, −156.86, −156.96, −156.98, −157.02, −157.04; MS ES (+) 543.20(M+H).

Example 11(S,S)-3-[2-(6,7-dichloro-1-oxo-1H-isoquinolin-2-yl)-butyrylamino]-4-oxo-5-(2,3,5,6-tetrafluoro-phenoxy)-pentanoicacid

Method Q: 5,6-Dichloro-3H-isobenzofuran-1-one

NaBH₄ (5.2 g) was added to a stirred solution of 4,5-dichlorophthalicanhydride (20 g) in anhydrous DMF (100 mL) at 0° C. under nitrogen insmall portions over 1 hour. The reaction was warmed to room temperaturefor a further 2 hours and poured into ice/1M HCl. The resultant whiteprecipitate (4,5-dichloro-2-hydroxymethyl-benzoic acid) was collected byfiltration and dried under vacuum. The precipitate was suspended intoluene (200 mL) with catalytic p-toluenesulfonic acid and heated toreflux under Dean-Stark conditions (precipitate dissolves on heating)for 18 hours. The reaction was cooled to room temperature and theresultant white precipitate collected by filtration to give thesub-title compound as a white solid (14.0 g, 75%). ¹H NMR (400 MHz,d₆-DMSO) δ 5.40 (2H, s), 8.05 (1H, s), 8.15 (1H, s) ppm.

Method R: 3-Bromo-5,6-dichloro-3H-isobenzofuran-1-one

A suspension of 5,6-Dichloro-3H-isobenzofuran-1-one (1.45 g),N-bromosuccinamide (1.27 g) and catalytic benzoyl peroxide in chloroform(30 mL) was heated to reflux for 1 hour. After cooling, the reactionmixture was washed with water, brine, dried (magnesium sulfate),filtered and concentrated to give the sub-title compound as a whitesolid (1.82 g, 91%). ¹H NMR (400 MHz, CDCl₃) δ 7.36 (1H, s), 7.77 (1H,s), 8.03 (1H, s) ppm.

Method S: 4,5-Dichloro-2-formyl-benzoic acid

A suspension of 3-bromo-5,6-dichloro-3H-isobenzofuran-1-one (2.0 g) in5% aqueous HCl (10 mL) and 80% aqueous dioxane (25 mL) was heated toreflux for 2 hours. The solvent was removed and the resulting residuere-dissolved in ethyl acetate, dried (magnesium sulfate) andconcentrated. The resultant yellow solid was recrystallized fromDCM/hexane to give the sub-title compound as a white solid (1.13 g,73%). ¹H NMR (400 MHz, CDCl₃) δ 6.66 (0.84H, s), 7.95 (0.16H, s), 8.05(0.84H), 8.12 (0.16H, s), 8.14 (0.84H, s), 8.41 (0.84H,), 10.41 (0.16H,s), 11.07 (0.16H, brd s) ppm.

(S,S)-3-[2-(6,7-dichloro-1-oxo-1H-isoquinolin-2-yl)-butyrylamino]-4-oxo-5-(2,3,5,6-tetrafluoro-phenoxy)-pentanoicacid

This compound was prepared using(S)-2-(6,7-dichloro-1-oxo-1H-isoquinolin-2-yl)-butyric acid (synthesizedfrom 4,5-dichloro-2-formyl-benzoic acid [prepared as described inmethods Q-S] using procedures similar to those described in methods A-E)and (3S)-3-amino-4-hydroxy-5-(2,3,5,6-tetrafluoro-phenoxy)-pentanoicacid tert-butyl ester (prepared as described in methods N-P) usingprocedures similar to those described in methods F, G and E. The titlecompound was isolated as a white solid (94% last step). IR (solid)1784.5, 1734.7, 1650.1, 1610.2, 1585.4, 1515.7, 1490.8, 1426.0, 1216.9,1172.1, 1092.5, 933.1 cm⁻¹; ¹H NMR (400 MHz, d₆-DMSO) δ 0.80 (3H, t),1.90-1.98 (1H, m), 2.04-2.12 (1H, m), 2.55-2.79 (2H, m), 4.56-4.71 (1H,m), 5.08-5.41 (3H, m), 6.67 (1H, d), 7.56-7.59 (2H, m), 8.07 (1H, brds), 8.25 (1H, d) 8.85-8.95 (1H, 2×d), 12.73 (1H, brd s) ppm; ¹⁹F NMR(376 MHz, d₆-DMSO) (proton decoupled) δ −140.93, −140.95, −140.99,−141.01, −141.04, −141.07, −141.10, −156.76, −156.79, −156.82, −156.85,−156.89, −156.91; MS ES (+) 577.14 (M+H).

Example 123-[(2S)-2-[6-(2-Chloro-phenoxy)-4-oxo-4H-quinazolin-3-yl)-butyrylamino]-5-fluoro-4-oxo-pentanoicacid

Method T: 2-Nitro-5-(2-chlorophenoxy)methyl benzoate

To a stirred solution of 5-chloro-2-nitrobenzoic acid methyl ester (1 g,4.6 mmol) in dimethylformamide (15 ml), was added potassium carbonate(0.96 g), and 2-chlorophenol (0.66 g). The reaction mixture was stirredat 90° C. for 16 hours. After this time the reaction mixture was pouredinto ethyl acetate (50 ml) and washed with water (50 ml), brine (50 ml),dried (MgSO₄), and evaporated. The residue was purified by flashchromatography (1:9 ethyl acetate: pet ether) to give the sub-titledcompound as a yellow oil (1.22 g, 85% yield). ¹H NMR (400 MHz, CDCl₃) δ8.05 (1H, m), 7.6-7.0 (6H, m), 3.95 (3H, s) ppm.

Method U: 2-Nitro-5-(2-chlorophenoxy)benzoic acid

To a stirred solution of 2-nitro-5-(2-chlorophenoxy)methyl benzoate(1.22 g) in THF (8 ml) was added lithium hydroxide (0.333 g) in water (2ml). The reaction mixture was stirred at ambient temperature for 16hours. After this time the reaction mixture was acidified with aqueous1M HCl. The aqueous layer was extracted with ethyl acetate (2×50 ml).The organics were combined and washed with saturated brine solution anddried (MgSO₄). The solvents were removed under vacuum to give thesub-title compound as an off-white solid (0.63 g, 54% yield). ¹H NMR(400 MHz, MeOD) δ 8.05-7.0 (7H, m) ppm.

3-[(2S)-2-[6-(2-Chloro-phenoxy)-4-oxo-4H-quinazolin-3-yl)-butyrylamino]-5-fluoro-4-oxo-pentanoicacid

This compound was prepared from2-(6-[2-chlorophenoxy]-4-oxo-4H-quinazoline-3-yl)-butyric acid,(synthesized from 5-chloro-2-nitrobenzoic acid methyl ester and2-chlorophenol as described in methods T-U, K-M and E) using proceduressimilar to those described in methods F, G and E. The title compound wasobtained as a white solid (TFA salt) (82%, last step). IR (solid)1783.38, 1721.55, 1664.48, 1550.32, 1498.00, 1474.22, 1264.94, 1193.60,1136.52, 1055.66 cm⁻¹; ¹H NMR (400 MHz, DMSO-d₆) δ 0.81 (3H, m),1.95-2.26 (2H, m), 2.50-2.96 (2H, m), 4.20-4.70 (1.5H, m), 5.04-5.46(2.5H, m), 7.26-7.39 (3H, m), 7.45 (1H, m), 7.57-7.70 (2H, m), 7.78 (1H,m), 8.35 (1H, m), 8.63-8.98 (1H, brm) ppm; ¹⁹F NMR (376 MHz, DMSO-d₆)(proton decoupled) δ −226.73, −226.78, −230.43, −230.90, −232.64,−232.55; MS ES (+) 490.40 (M+H).

Example 133-[(2S)-2-[6-(3-Chlorophenoxy)-4-oxo-4H-quinazolin-3-yl)-butyrylamino]-5-fluoro-4-oxo-pentanoicacid

This compound was prepared from2-(6-[3-chlorophenoxy]-4-oxo-4H-quinazoline-3-yl)-butyric acid,(synthesized from 5-chloro-2-nitrobenzoic acid methyl ester and3-chlorophenol using methods similar to those described in T-U, K-M andE) using procedures similar to those described in methods F, G and E.The product was obtained as a white solid (TFA salt) (85%, last step).IR (solid) 1716.79, 1673.99, 1583.62, 1469.46, 1279.21, 1198.35, 1136.52cm⁻¹; ¹H NMR (400 MHz, DMSO-d₆) δ 0.75-0.9 (3H, m), 1.95-2.2 (2H, m),2.5-2.9 (2H, m), 4.2-4.7 (2H, m), 5.05-5.45 (2H, m), 7.1 (1H, m), 7.25(1H, m), 7.3 (1H, m), 7.45 (1H, m), 7.55 (1H, m), 7.6 (1H, m), 7.8 (1H,m), 8.35 (1H, m), and 8.65-9.0 (1H, m) ppm; ¹³C NMR (100 MHz, DMSO-d₆) δ202.6, 202.5, 173.1, 172.0, 172.0, 170.0, 169.8, 169.8, 160.2, 160.1,158.8, 157.4, 155.3, 144.1, 134.5, 132.1, 130.1, 126.6, 124.6, 122.6,122.5, 119.7, 118.2, 113.7, 85.3, 85.2, 83.5, 83.4, 82.9, 58.2, 57.8,57.5, 53.0, 52.3, 52.2, 47.8, 34.6, 33.0, 24.4, 23.8 and 10.66; ¹⁹F NMR(376 MHz, DMSO-d₆) (proton decoupled) δ −226.72, −226.78, −230.90 and−232.65; MS ES (+) 490.25 (M+H).

Example 145-Fluoro-3-{(2S)-2[6-(3-fluorophenoxy)-4-oxo-4H-quinazolin-3-yl]-butyrylamino}-4-oxo-pentanoicacid

This compound was prepared from2-[6-(3-fluorophenoxy)-4-oxo-4H-quinazolin-3-yl]-butyric acid,(synthesized from 5-chloro-2-nitrobenzoic acid methyl ester and3-fluorophenol using methods similar to those described in T-U, K-M andE) using procedures similar to those described in methods F, G and E.The product was obtained as a white solid (TFA salt) (95%, last step).IR (solid) 1598.20, 1274.17, 1178.86, 1159.80, 1116.92 and 954.90 cm⁻¹;¹H NMR (400 MHz, DMSO-d₆) δ 0.8-0.95 (3H, m), 1.95-2.2 (2H, m),2.55-2.95 (2H, m), 4.2-4.7 (2H, m), 5.05-5.5 (2H, m), 6.95 (1H, m), 7.05(1H, m), 7.45 (1H, m), 7.55 (1H, m), 7.6 (1H, m), 7.8 (1H, m), 8.4 (1H,m), and 8.65-9.0 (1H, m) ppm; ¹³C NMR (100 MHz, DMSO-d₆) δ 172.0, 169.9,169.8, 164.5, 162.1, 160.2, 155.3, 144.1, 132.0, 131.9, 130.0, 126.9,122.6, 115.5, 113.7, 111.5, 111.3, 107.4, 107.1, 85.3, 58.2, 57.8, 52.4,52.2, 34.6, 23.8, and 10.7; ¹⁹F NMR (376 MHz, DMSO-d₆) (protondecoupled) δ −110.84, −226.73, −226.79, −230.41, −230.91, −232.64 and−232.66; MS ES (+) 474.29 (M+H).

Example 153-{(2S)-2-[6-(2,4-Dichloro-phenoxy)-4-oxo-4H-quinazolin-3-yl]-butyrylamino}-5-fluoro-4-oxo-pentanoicacid

This compound was prepared from2-[6-(2,4-dichlorophenoxy)-4-oxo-4H-quinazolin-3-yl]-butyric acid,(synthesized from 2-nitro-5-2,4-dichlorophenoxybenzoic acid methyl esterusing methods similar to those described in U, K-M and E) usingprocedures similar to those described in methods F, G and E. The productwas obtained as a white solid (TFA salt)(96%, last step). IR (solid)1778.63, 1716.79, 1669.23, 1493.25, 1469.46, 1264.94, 1198.35, 1150.79,1098.47 and 1055.66 cm⁻¹; ¹H NMR (400 MHz, DMSO-d₆) δ 0.60-1.05 (3H, m),1.94-2.26 (2H, m), 2.26-2.95 (2H, m), 4.15-4.75 (2H, m), 4.76-5.95 (2H,m), 7.25-7.45 (2H, m), 7.50 (1H, m), 7.65 (1H, m), 7,80 (1H, m), 7.87(1H, m), 8.36 (1H, s), 8.60-9.01 (1H, brm) ppm; ¹⁹F NMR (376 MHz,DMSO-d₆) (proton decoupled) δ −226.72, −226.77, −230.41, −230.88,−232.65; MS ES (+) 524.34 (M+H).

Example 165-Fluoro-4-oxo-3-[(2S)-2-(4-oxo-6-phenylsulfanyl-4H-quinazolin-3-yl)-butyrylamino]-pentanoicacid

This compound was prepared from(S)-2(4-oxo-6-phenylsulfanyl-4H-quinazolin-3-yl)-butyric acid,(synthesized from 5-chloro-2-nitrobenzoic acid methyl ester andthiophenol using methods similar to those described in T-U, K-M and E)using procedures similar to those described in methods F, G and E. Theproduct was obtained as a white solid (TFA salt)(81%, last step). IR(solid) 1791.61, 1720.53, 1668.40, 1602.06, 1545.20, 1474.12, 1194.53,1142.41 and 1057.11 cm⁻¹; ¹H NMR (400 MHz, DMSO-d₆) δ 9.6-8.6 (1H, m),8.3 (1H, s), 7.85 (1H, s), 7.75-7.6 (2H, m), 7.45-7.35 (5H, m), 5.4-5.0(2H, m), 4.6-4.15 (2H, m), 2.85-2.35 (2H, m), 2.2-1.95 (2H, m) and0.85-0.7 (3H, m) ppm; ¹³C NMR (100 MHz, DMSO-d₆) δ 202.6, 202.5, 173.1,172.0, 172.0, 169.9, 169.8, 160.0, 146.5, 136.1, 135.4, 133.6, 133.3,130.3, 128.8, 128.7, 126.7, 126.6, 122.2, 122.2, 104.0, 85.3, 85.2,83.5, 83.4, 58.3, 57.9, 57.4, 52.3, 52.2, 47.8, 34.6, 33.0, 24.4, 23.9,23.8 and 10.6; ¹⁹F NMR (376 MHz, DMSO-d₆) (proton decoupled) δ −226.72,−226.78, −230.41, −231.92, −232.65 and −232.68; MS ES (+) 472.27 (M+H).

Example 175-Fluoro-3{(2S)-2[6-(3-fluoro-phenylsulfanyl)-4-oxo-4H-quinazolin-3-yl]-butyrylamino}-4-oxo-pentanoicacid

This compound was prepared from(S)-2[6-(3-fluoro-phenylsulfanyl)-4-oxo-4H-quinazolin-3-yl]-butyric acid(synthesized from 5-chloro-2-nitrobenzoic acid methyl ester and3-fluorothiophenol using methods similar to those described in T-U, K-Mand E) using procedures similar to those described in methods F, G andE. The product was obtained as a white solid (TFA salt)(95%, last step).IR (solid) 1779.2, 1722.0, 1660.1, 1602.9, 1574.3, 1474.3, 1178.8 and1059.7 cm⁻¹; ¹H NMR (400 MHz, DMSO-d₆) δ 0.80 (3H, m), 1.92-2.25 (2H,m), 2.41-2.92 (2H, m), 4.20-4.85 (1.5H, m), 5.01-5.50 (2.5H, m),7.12-7.27 (3H, m), 7.40-7.50 (1H, m), 7.70-7.86 (2H, m), 8.03 (1H, m),8.43 (1H, m), 8.65-9.02 (1H, brm) ppm; ¹⁹F NMR (376 MHz, DMSO-d₆)(proton decoupled) δ −111.83, −226.73, −226.79, −230.40, −230.91,−232.65, −232.67; MS ES (+) 490.35 (M+H).

Example 18(S)-3-[2-(7-chloro-1-oxo-1H-isoquinolin-2-yl)-3-methylbutyrylamino]-5-fluoro-4-oxo-pentanoicacid

This was prepared using procedures similar to those described in methodsA-G (S-valine tert-butyl ester was used in method C). The product wasisolated as a white solid (91% last step). IR (solid) 1777.5, 1644.4,1608.5, 1588.1, 1541.9, 1198.9, 1168.1, 1045.2, 830.2 cm⁻¹; ¹H NMR (400MHz, d₆-DMSO) δ 0.70 (3H, m), 0.89-1.14 (3H, m), 2.31 (1H, m), 2.45-2.98(2H, m), 4.05-4.75 (1.6H, m), (2.4H, m), 6.70 (1H, m), 7.60-7.95 (3H,m), 8.18 (1H, m), 8.75-9.21 (1H, m) ppm; ¹⁹F NMR (376 MHz, d₆-DMSO)(proton decoupled) δ −226.28, −226.71, −230.58, −230.64, −231.75,−232.32; MS ES (+) 411.36 (M+H).

Example 19(S,S)-3-[2-(7-trifluoromethyl-1-oxo-1H-isoquinolin-2-yl)-butyrylamino]-4-oxo-5-(2,3,5,6-tetrafluoro-phenoxy)-pentanoicacid

This compound was prepared using(S)-2-(7-trifluoromethyl-1-oxo-1H-isoquinolin-2-yl)-butyric acid(prepared as described in methods A-E, see example 2) and(3S)-3-amino-4-hydroxy-5-(2,3,5,6-tetrafluoro-phenoxy)-pentanoic acidtert-butyl ester (prepared as described in methods N-P) using proceduressimilar to those described in methods F, G and E. The product wasisolated as a white solid (90% last step). IR (solid) 1791.6, 1649.5,1516.8, 1493.1, 1322.5 cm⁻¹; ¹H NMR (400 MHz, d₆-DMSO) δ 0.85 (3H, t),1.90-2.20 (2H, 2×m), 2.50-2.90 (2H, 2×m), 4.70 (1H, m), 5.25 (2H, dd),5.45 (1H, m), 6.80 (1H, d), 7.65 (1H, m), 7.70 (1H, d), 7.95 (1H, d),8.05 (1H, d), 8.45 (1H, s), 9.00 (1H, d) ppm; ¹⁹F NMR (376 MHz, d₆-DMSO)(proton decoupled) δ −56.60, −70.15, −136.38, −152.08.

Example 20(S,S)-3-[2-(7-trifluoromethyl-4-oxo-4H-quinazolin-3-yl)-butyrylamino]-4-oxo-5-(2,3,5,6-tetrafluoro-phenoxy)pentanoicacid

Method V: (S)-2-(2-Benzylamino-5-trifluoromethyl-benzoylamino)-butyricacid tert-butyl ester

To a solution of (S)-2-(2-chloro-5-trifluoromethyl-benzoylamino)-butyricacid tert-butyl ester (prepared from 2-chloro5-trifluoromethylbenzoicacid and (S)-2-aminobutyric acid, tert butyl ester in a proceduresimilar to that described in method F) (3 g) and benzylamine (1.1 ml) inN-methylpyrrolidine (40 ml) was added potassium carbonate (1.6 g),copper (30 mg) and copper (I) bromide (15 mg). The mixture was heated at160° C. for 3 hours then cooled to room temp. and diluted with saturatedammonium chloride solution. The mixture was extracted with ethyl acetateand the organic solution dried (magnesium sulfate), filtered andconcentrated. The residue was purified on silica gel, eluting with 15%ethyl acetate/hexane. The sub-titled product was obtained as a whitesolid (950 mg, 27%). ¹H NMR (400 MHz, CDCl₃) δ 1.04 (3H, t), 1.50 (9H,s), 2.86 (1H, m), 2.02 (1H, m), 4.50 (2H, d), 4.65 (1H, m), 6.69 (2H,m), 7.21-7.70 (7H, m), 8.42 (1H, m) ppm; MS ES (−) 435.32 (M−H).

(S)-2-(6-trifluoromethyl-4-oxo-4H-quinazoline-3-yl)-butyric acid

This compound was prepared from(S)-2-(2-benzylamino-5-trifluoromethyl-benzoylamino)-butyric acidtert-butyl ester using procedures similar to those described in methodsP, M and E. The sub-title compound was as a white solid. ¹H NMR (400MHz, d₆-DMSO) δ 0.88 (3H, t), 2.22 (2H, m), 5.15 (1H, m), 7.95 (1H, m),8.20 (1H, m), 8.40 (1H, s), 8.60 (1H, s) ppm; MS ES (+) 301.15 (M+H).

(S,S)-3-[2-(7-trifluoromethyl-4-oxo-4H-quinazolin-3-yl)-butyrylamino]-4-oxo-5-(2,3,5,6-tetrafluoro-phenoxy)pentanoicacid

This compound was prepared from2-(6-trifluoromethyl-4-oxo-4H-quinazoline-3-yl)-butyric acid and(3S)-3-amino-4-hydroxy-5-(2,3,5,6-tetrafluoro-phenoxy)-pentanoic acidtert-butyl ester (prepared as described in methods N-P) using proceduressimilar to those described in methods F, G and E. The titled compoundwas obtained as a white solid (TFA salt) (90% last step). IR (solid)1690, 1518, 1490, 1317, 1254, 1170, 1128 cm⁻¹; ¹H NMR (400 MHz, d₆-DMSO)δ 0.8-0.9 (3H, m), 2.05-2.2 (2H, m), 2.57-2.80 (2H, m), 4.55-4.62 (2H,m), 5.20-5.35 (3H, m), 7.50-7.62 (1H, m), 7.95 (1H, d), 8.20 (1H, d),8.38 (1H, m), 8.52 (1H, s), 9.04 (1H, d) ppm; MS ES (+) 578.2 (M+H).

Example 21(S)-3-[2-(7-phenyl-1-oxo-1H-isoquinolin-2-yl)-butyrylamino]-5-fluoro-4-oxo-pentanoicacid

Method W: (S)-2-(7-phenyl-1-oxo-1H-isoquinolin-2-yl)-butyric acid

To a solution of (S)-2-(7-chloro-1-oxo-1H-isoquinolin-2-yl)-butyric acid(prepared as described in methods A-E) (200 mg) and phenyl boronic acid(276 mg) in THF (2 ml) was added palladium (II) acetate (1.7 mg),2-(di-tert-butylphosphino)biphenyl (4.5 mg) and potassium fluoride (262mg). The resulting mixture was heated at reflux for 18 hours, thencooled and diluted with 1M HCl (5 ml) and ethyl acetate (15 ml). Theorganic phase was separated and the aqueous extracted with ethyl acetate(2×15 ml). The combined organics were dried (magnesium sulfate),filtered and concentrated. The residue was purified through a plug ofsilica gel, then further purified by radial chromatography eluting with1% acetic acid in ethyl acetate. This gave the subtitled product as abrown solid (153 mg, 66%). ¹H NMR (400 MHz, CDCl₃) δ 1.00 (3H, t), 2.15(1H, m), 2.40 (1H, m), 5.37 (1H, m), 6.70 (1H, d), 7.15 (1H, d), 7.45(1H, m), 7.55 (2H, m), 7.68 (1H, m), 7.75 (2H, m), 7.99 (1H, d), 8.70(1H, s) ppm.

(S)-3-[2-(7-phenyl-1-oxo-1H-isoquinolin-2-yl)-butyrylamino]-5-fluoro-4-oxo-pentanoicacid

This compound was prepared from(S)-2-(7-phenyl-1-oxo-1H-isoquinolin-2-yl)-butyric acid using proceduressimilar to those described in methods F, G and E. The titled compoundwas obtained as a white solid (58% last step). ¹H NMR (400 MHz, d₆-DMSO)δ 8.95-8.5 (1H, m), 8.45 (1H, s), 8.05 (1H, m), 7.8-7.7 (2H, m),7.65-7.35 (4H, m), 6.7-6.65 (1H, m), 5.6-5.0 (2.4H, m), 4.7-4.2 91.6H,m), 2.9-2.3 (H, m), 2.15-1.85 (2H, m), 0.8-0.7 (3H, m) ppm; ¹³C NMR (100MHz, DMSO-d₆) δ 202.8, 202.6, 173.2. 172.0, 170.6, 170.5, 161.7, 139.7,138.7, 138.7, 136.1, 136.1, 131.0, 130.9, 130.8, 127.3, 125.8, 125.8,125.7, 125.1, 105.1, 105.0, 104.8, 85.3, 85.2, 83.5, 83.4, 58.9, 58.5,58.0, 52.3, 52.2, 47.6, 34.7, 34.6, 33.0, 32.9, 24.6, 24.1, 23.9; ¹⁹FNMR (376 MHz, d₆-DMSO) (proton decoupled) δ −226.68, −226.72, −230.50,−231.17, −232.60, −232.66; MS ES (+) 439.0 (M+H).

Example 225-Fluoro-4-oxo-3-[(2S)-2-(4-oxo-6-propylsulfanyl-4H-quinazolin-3-yl)-butyrylamino]-pentanoicacid

(S)-2-(4-oxo-6-propylsulfanyl-4H-quinazoline-3-yl)-butyric acid,tert-butyl ester

This compound was prepared from 5-chloro-2-nitrobenzoic acid methylester and propanethiol using procedures similar to those described inmethods T-U and K-M. The sub-title compound was as a solid. ¹H NMR (400MHz, CDCl₃) δ 1.00 (3H, t), 1.10 (3H, t), 1.51 (9H, s), 1.75 (2H, m),2.01 (1H, m), 2,31 (1H, m), 3.03 (2H, m), 5.30 (1H, m), 7.60-7.70 (2H,m), 8.05 (1H, s), 8.13 (1H, s) ppm; MS ES (+) 363.29 (M+H).

5-Fluoro-4-oxo-3-[(2S)-2-(4-oxo-6-propylsulfanyl-4H-quinazolin-3-yl)-butyrylamino]-pentanoicacid

This compound was prepared from(S)-2-(4-oxo-6-propylsulfanyl-4H-quinazoline-3-yl)-butyric acid(synthesized from the tert-butyl ester using TFA/DCM) using a proceduresimilar to that described in method F. The resulting alcohol wasoxidized using tetra-n-propylammoniumperruthenate(VII)/N-methylmorpholine-N-oxide (Synthesis, 639 (1994)) andthe tert-butyl ester deprotected as described in method E. The titlecompound was obtained as a white solid (90% last step) (TFA salt). IR(solid) 2963.9, 1786.2, 1739.4, 1666.8, 1195.2 cm⁻¹; ¹H NMR (400 MHz,CDCl₃) δ 1.02 (3H, t), 1.08 (3H, t), 1.70-1.80 (2H, m), 2.02-2.09 (1H,m), 2.29-2.36 (1H, m), 2.72-2.92 (1H, m), 3.00-3.15 (1H, m), 3.03 (2H,t), 4.50-5.20 (3H, m), 5.40-5.60 (1H, m), 7.69-7.75 (2H, m), 8.06-8.07(1H, m), 8.60-8.64 (1H, m) ppm; ¹⁹F NMR (376 MHz, CDCl₃) (protondecoupled) −76.23, −231.5; MS ES (+) 436.3 (M+H)

Example 23 Enzyme Assays

The assays for caspase inhibition are based on the cleavage of afluorogenic substrate by recombinant, purified human Caspases −1, −3, −7or −8. The assays are run in essentially the same way as those reportedby Garcia-Calvo et al., J. Biol. Chem. 273, 32608-32613 (1998), using asubstrate specific for each enzyme. The substrate for Caspase-1 isAcetyl-Tyr-Val-Ala-Asp-amino-4-methylcoumarin and the substrate forCaspases −3, −7 and −8 is Acetyl-Asp-Glu-Val-Asp-amino-4-methylcoumarinboth as disclosed in Garcia-Calvo et al., J. Biol. Chem. 273,32608-32613 (1998).

The observed rate of enzyme inactivation at a particular inhibitorconcentration, k_(obs), is computed by direct fits of the data to theequation derived by Thornberry et al., Biochemistry, 33, 3943-3939(1994) using a nonlinear least-squares analysis computer program (PRISM2.0; GraphPad software). To obtain the second order rate constant,k_(inact), k_(obs) values are plotted against their respective inhibitorconcentrations and k_(inact) values are subsequently calculated bycomputerized linear regression.

Table 2 shows inhibition of caspases −1, −3, and −8 activity forselected compounds of this invention as determined by the above method.Compounds with k_(inact)(M⁻¹s⁻¹)>500000 are listed as “A”. Compoundswith k_(inact)(M⁻¹s⁻¹) between 100000 and 500000 are listed as “B”.Compounds with k_(inact)(M⁻¹s⁻¹)<100000 are listed as “C”.

TABLE 2 Inhibition of Caspases-1, -3, and -8 Compound k_(inact) NumberCaspase-1 Caspase-3 Caspase-8 1 A A B 2 A A A 3 B B C 4 B B C 5 A A B 6B A C 7 C B C 8 B B C 9 B B C 10 A A C 11 A A C 12 A A A 13 B B A 14 A AB 15 B B A 16 A A A 17 A A A 18 B B C 19 A A C 20 A A C 21 A A A 22 A AA

Example 24 Inhibition of IL-1β Secretion from Whole Blood

Human blood is freshly drawn from healthy donors and diluted 1:2 in PBS.To 500 μl of diluted blood 50 ml of prediluted test compound in RPMImedium and 10 ml LPS (5 ng/ml final concentration on the plate) areadded (LPS, Serotype 0111:B4, Sigma L3012). After stimulation for 18hours supernatants are collected and assayed for IL-1β levels using theappropriate ELISA kit (R&D systems).

Table 3 below shows inhibition of IL-1β secretion from human whole bloodfor selected compounds of this invention as determined by the abovemethods. Compounds with an IC₅₀>10 μM are listed as “A”. Compounds withan IC₅₀ between 1 μM and 10 uM are listed as “B”. Compounds with anIC₅₀<1 μM are listed as “C”.

TABLE 3 Inhibition of IL-1β secretion Compound Number IC₅₀ (μm) 1 C 2 C3 B 4 B 5 B 6 B 7 A 8 B 9 A 10 B 11 A 12 B 13 B 14 C 15 B 16 B 17 B 18 B19 B 20 A 21 B 22 C

Example 25 Hypoxia-Induced Apoptosis of Rat Cortical Neurons

Cortical neurons are dissociated from Wistar rat embryos (E17) by amodification of the procedure of Rogers et al., Brain Res. Bulletin,44:131 (1998). Briefly, cerebral cortices are isolated aseptically from15-20 Wistar rat embryos. A cell suspension is prepared by mincing thecerebral cortices and digesting them with papain. Cells are washed withovomucoid enzyme inhibitor and DNaseI and plated onto Poly-D lysinecoated plates in high glucose DMEM containing 10% heat-inactivated fetalcalf serum, L-glutamine, penicillin and streptomycin. The yield ofneurons is 10×7 per embryo and they are 80-90% viable as assessed byTrypan blue exclusion.

The neurons are cultured in complete medium at 37° C. in a normalatmosphere for 48 hours prior to the hypoxia experiments. For hypoxia,the normal cell medium is replaced by oxygen-depleted serum-free medium.Cells are incubated in an atmosphere of 95% N₂/5% CO₂ for variouslengths of time. Compounds are dissolved in DMSO at 100 mM then dilutedin medium and added to the culture from time=0. The level of apoptosisis measured using a Cell Death Detection ELISA kit (Roche) which detectsDNA fragmentation. Plates are read at 405 nm. Controls included cellscultured in aerobic conditions in serum-containing medium (+serum) andcells cultured in aerobic conditions in serum-deprived medium (−serum).

Table 4 shows the results of the activity of selected compounds of thisinvention in the Hypoxia-induced apoptosis of rat cortical neurons.Compounds with an IC₅₀>0.5 μM are listed as “A”. Compounds with an IC₅₀between 0.1 μM and 0.5 uM are listed as “B”. Compounds with an IC₅₀<0.1μM are listed as “C”.

TABLE 4 Activity in Hypoxia-induced Apoptosis Assay Compound Number IC₅₀(μm) 1 B 2 A 3 B 4 A 5 B 6 B 7 A 8 B 9 B 10 C 11 B 12 A 13 B 14 A 15 B16 B 17 B 18 B 19 C 20 B 21 B 22 A

While we have described a number of embodiments of this invention, it isapparent that our basic examples may be altered to provide otherembodiments that utilize the compounds and methods of this invention.Therefore, it will be appreciated that the scope of this invention is tobe defined by the appended claims rather than by the specificembodiments that have been represented by way of example above.

1. A compound of formula I:

X is N; Y is halo, trifluorophenoxy, or tetrafluorophenoxy; R² is C₁₋₆straight chained or branched alkyl; R³ is hydrogen, halo, OCF₃, CN, orCF₃; and R⁴ is hydrogen, halo, OCF₃, SR, CN, CF₃, Ar, or T-Ar; wherein:T is O or S; R is a C₁₋₆ straight chained or branched alkyl; Ar is aphenyl ring optionally substituted with 1-3 groups selected from halo,CH₃, CF₃, CN, OMe, OCF₃, and NR⁵R⁶; and R⁵ and R⁶ each is independentlyH or C₁₋₆ straight chained or branched alkyl, or R⁵ and R⁶, takentogether, form a 5-7 membered ring optionally containing up to 3heteroatoms selected from O, S, NH, and N(C₁₋₆-straight chained orbranched alkyl); provided that when Y is halo, then both, R³ and R⁴, arenot simultaneously hydrogen.
 2. The compound according to claim 1,wherein R² is ethyl, n-propyl, or isopropyl.
 3. The compound accordingto claim 2, wherein Y is F, trifluorophenoxy, or tetrafluorophenoxy.4-8. (canceled)
 9. The compound according to claim 1, having the formulaIB:

wherein: X is N; R² is ethyl, n-propyl, or isopropyl; R³ and R⁴ are eachindependently hydrogen, halo, OCF₃, CN, or CF₃; and Ar² istrifluorophenyl or tetrafluorophenyl.
 10. The compound according toclaim 9, wherein Ar² is 2,3,5,6-tetrafluorophenyl.
 11. The compoundaccording to claim 9, wherein R² is ethyl.
 12. (canceled)
 13. Thecompound according to claim 9, wherein R⁴ is Cl or CF₃; Ar² is2,3,5,6-tetrafluorophenyl; and R² is ethyl.
 14. The compound accordingto claim 13 wherein R³ is H, and R⁴ is F, Cl, or CF₃.
 15. The compoundaccording to claim 1, having formula IC:

wherein: R² is ethyl, n-propyl, or isopropyl; R³ is hydrogen, halo,OCF₃, CN, or CF₃; R⁴ is halo, OCF₃, CN, CF₃, SR, or T-Ar; T is O or S; Ris a C₁₋₆ straight chained or branched alkyl; Ar is a phenyl ringoptionally substituted with 1-3 groups selected from halo, CH₃, CF₃, CN,OMe, OCF₃, and NR⁵R⁶; and R⁵ and R⁶ each is independently H or C₁₋₆straight chained or branched alkyl, or R⁵ and R⁶, taken together, form a5-7 membered ring optionally containing up to 3 heteroatoms selectedfrom O, S, NH, and N(C₁₋₆-straight chained or branched alkyl).
 16. Thecompound according to claim 15, wherein R² is ethyl.
 17. The compoundaccording to claim 16, wherein R³ is hydrogen.
 18. The compoundaccording to claim 17, wherein T is O.
 19. The compound according toclaim 18, wherein R⁴ is T-Ar.
 20. The compound of claim 1, selectedfrom:


21. A pharmaceutical composition comprising: a) a compound according toclaim 1; and b) a pharmaceutically acceptable carrier, adjuvant orvehicle. 22-30. (canceled)
 31. A method of preparing a compound offormula I,

said method comprising: reacting an acid or acid derivative of formulaII,

with an amino alcohol of formula B, to provide a compound of formulaIII,

converting intermediate III to compound I, wherein; X is N; Y is halo,trifluorophenoxy, or tetrafluorophenoxy; R² is a C₁₋₆ straight chainedor branched alkyl; R³ is hydrogen, halo, OCF₃, CN, or CF₃; and R⁴ ishydrogen, halo, OCF₃, SR, CN, CF₃, Ar, or T-Ar; wherein: T is O or S; Ris a C₁₋₆ straight chained or branched alkyl; Ar is a phenyl ringoptionally substituted with 1-3 groups selected from halo, CH₃, CF₃, CN,OMe, OCF₃, and NR⁵R⁶; R⁵ and R⁶ each is independently H or C₁₋₆ straightchained or branched alkyl, or R⁵ and R⁶, taken together, form a 5-7membered ring optionally containing up to 3 heteroatoms selected from O,S, NH, and N(C₁₋₆-straight chained or branched alkyl); and R⁷ is asuitable protecting group; provided that when Y is halo, then both, R³and R⁴, are not simultaneously hydrogen.
 32. A compound of formula IIA:

wherein; X is N; R² is a C₁₋₆ straight chained or branched alkyl; R³ ishydrogen, halo, OCF₃, CN, or CF₃; and R⁴ is hydrogen, halo, OCF₃, SR,CN, CF₃, Ar, or T-Ar; wherein: T is O or S; R is a C₁₋₆ straight chainedor branched alkyl; Ar is a phenyl ring optionally substituted with 1-3groups selected from halo, CH₃, CF₃, CN, OMe, OCF₃, and NR⁵R⁶; and R⁵and R⁶ each is independently H or C₁₋₆ straight chained or branchedalkyl, or R⁵ and R⁶, taken together, form a 5-7 membered ring optionallycontaining up to 3 heteroatoms selected from O, S, NH, andN(C₁₋₆-straight chained or branched alkyl).
 33. The compound accordingto claim 31 or 32 wherein R² is ethyl or isopropyl.